Vaccine and methods for detecting and preventing filariasis

ABSTRACT

The present invention is a multivalent immunogenic composition for immunizing an animal against filariasis. In some embodiments, the antigens of the multivalent immunogenic composition are protein-based, DNA-based, or a combination thereof. This invention also provides a method and kit for detecting a filarial nematode and determining vaccine efficacy.

INTRODUCTION

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/116,203, filed Aug. 29, 2018, which is acontinuation of U.S. patent application Ser. No. 14/798,945, filed Jul.14, 2015, now U.S. Pat. No. 10,072,054 B2, which is continuation-in-partapplication of U.S. patent application Ser. No. 13/885,168, filed May23, 2013, now abandoned, which is the National Stage of InternationalApplication No. PCT/US2011/059501, filed Nov. 7, 2011; which claims thebenefit of priority from U.S. Provisional Application Ser. No.61/413,681, filed Nov. 15, 2010; U.S. Provisional Application Ser. No.61/449,954, filed Mar. 7, 2011; and U.S. Provisional Application Ser.No. 61/522,079, filed Aug. 10, 2011, and also claims the benefit ofpriority from U.S. Provisional Application Ser. No. 62/805,366, filedFeb. 14, 2019 and U.S. Provisional Application Ser. No. 62/951,341,filed Dec. 20, 2019, the contents of which are incorporated herein byreference in their entireties.

This invention was made with government support under contract numbersAI064745 and AI116441 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Lymphatic filariasis caused by the filarial nematodes Wuchereriabancrofti, Brugia malayi, and Brugia timori, affects more than 120million people worldwide (WHO (1992) World Health Organ. Tech. Rep. Ser.821:1-71). Mass drug administration program by the World HealthOrganization, is significantly reducing the incidence rate of lymphaticfilariasis in many parts of the world (Hotez (2009) Clin. Pharmacol.Ther. 85(6):659-64). Nevertheless, lack of effectiveness to the massdrug administration has been reported from several endemic regionsmainly due to noncompliance (Babu & (2008) Trans. R. Soc. Trop. Med.Hyg. 102(12):1207-13; El-Setouhy, et al. (2007) Am. J. Trop. Med. Hyg.77(6):1069-73). In addition, drug resistance has been reported to atleast one of the drugs in the mass drug combination (Horton (2009) Ann.Trop. Med. Parasitol. 103(1):S33-40; Schwab, et al. (2007) Parasitology134(Pt 7):1025-40). Since yearly administration of the mass drugs isrequired for effective control, there is an alarming concern forselecting drug resistant parasites. Therefore, there is an immediateneed for a multipronged approach in controlling this mosquito borneinfection.

As with lymphatic filariasis, treatment of dirofilariasis (heartwormdisease) in canids and felids has included the use of macrolide agentssuch as ivermectin, milbemycin oxime, moxidectin and selamectin, whichprevent larval development during the first 2 months after infection.However, these agents must be administered monthly for effectiveness andcan be very expensive to a pet owner.

Vaccination is one strategy for controlling these infections and severalsubunit candidate vaccine antigens have been tested in laboratoryanimals with variable results (Bottazzi, et al. (2006) Expert Rev.Vaccines 5(2):189-98; Chenthamarakshan, et al. (1995) Parasite Immunol.17(6):277-85; Dissanayake, et al. (1995) Am. J. Trop. Med. Hyg.53(3):289-94; Li, et al. (1993) J. Immunol. 150(5):1881-5; Maizels, etal. (2001) Int. J. Parasitol. 31(9):889-98; Thirugnanam, et al. (2007)Exp. Parasitol. 116(4):483-91; Veerapathran, et al. (2009) PLoS Negl.Trop. Dis. 3(6):e457). Lymphatic filariasis is a multicellular organismwith complex life cycle and produce large array of host modulatorymolecules. Thus, fighting against this infection with a single antigenvaccine can be difficult. By screening a phage display cDNA expressionlibrary of the B. malayi parasite with sera from immune individuals,several potential vaccine candidates were identified (Gnanasekar, et al.(2004) Infect. Immun. 72(8):4707-15). However, a varying degree ofprotection was achieved with each of the candidate vaccine antigens whengiven as a DNA, protein or prime boost vaccine (Veerapathran, et al.(2009) supra).

SUMMARY OF THE INVENTION

The present invention is a multivalent immunogenic composition composedof two or more antigens from one or more filarial nematodes, e.g.,Brugia malayi, Wuchereria bancroft, Onchocerca volvulus, Loa loa, Brugiatimori or Dirofilaria immitis. In some embodiments, the antigens areprotein-based, DNA-based, or a combination thereof. In otherembodiments, the antigens include an Abundant Larval Transcript,Tetraspanin, Small heat shock protein (HSP) 12.6, Thioredoxin Peroxidase2, or fragments thereof, in particular an Abundant Larval Transcript ofSEQ ID NO:121 or SEQ ID NO:122; a Small heat shock protein 12.6 of SEQID NO:81 or SEQ ID NO:123; a Tetraspanin of SEQ ID NO:82; or aThioredoxin Peroxidase 2 of SEQ ID NO:83 or SEQ ID NO:124. In certainaspects, the antigens are covalently attached. This invention alsoprovides a recombinant vector harboring nucleic acids encoding themultivalent immunogenic composition, a recombinant host cell harboringthe recombinant vector, and the inclusion of an adjuvant in themultivalent immunogenic composition. Methods for inducing a protectiveimmune response in a subject and immunizing an animal against filariasisor dirofilariasis are also provided. In some embodiments of thesemethods, the multivalent immunogenic composition is administered with anadjuvant, e.g., in one or more additional doses by subcutaneous orintramuscular injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the titer of anti-BmHSP and anti-BmALT2 IgG antibodies inthe sera of vaccinated mice. 6-week-old balb/c mice were immunized usinga prime boost approach with a monovalent immunogenic composition (Bmhspprime and rBmHSP boost or Bmalt2 prime and rBmALT2 boost) andmultivalent immunogenic composition (Bmhsp/Bmalt2 prime and rBmHSP andrBmALT2 boost). Titer of IgG antibodies were measured in the sera usingan indirect ELISA. The data presented is the antibody titer 2 weeksafter the last booster. Results show that both bivalent and multivalentimmunogenic compositions induce significant IgG antibodies against eachof the component antigens. The findings also show that the antigens inthe monovalent and multivalent formulations act synergistically inboosting the immune responses. N=5. Statistically significant **p<0.001, * p<0.05. Values represented are mean±SD.

FIGS. 2A-2B show the number of IL-4 (FIG. 2A) and IFN-γ (FIG. 2B)secreting cells in the spleen of mice vaccinated with monovalent (BmHSPor BmALT2) or multivalent immunogenic composition. An ELISPOT assay wasperformed after stimulating the cells with rBmHSP or rBmALT (1 μg/ml).Single cell preparations of spleen cells were stimulated with respectiveantigens for 48 hours and spot forming cells were counted. Results showthat both monovalent and multivalent immunogenic compositions promotedIL-4 secreting cells. Multivalent vaccination induced the higher numberof IL-4 producing cells than controls. IFN-γ producing cells werecomparatively low. These findings further confirm that BmHSP and BmALT2synergistically boost the immune responses in vaccinated animalsfollowing a multivalent vaccination. N=5. Results are expressed as meannumber of spot forming units per 3×10⁶ cells±SD.

FIG. 3 shows the degree of protection conferred by a multivalentimmunogenic composition in a mouse model. Balb/c strain of mice wereimmunized with HAT (HSP/ALT2/TSP) hybrid DNA, with recombinant HATprotein or a combination of both using a prime boost approach. HAThybrid DNA was used for priming. Two weeks following the priming, micewere boosted with HAT hybrid protein. Another group of mice wereimmunized with HAT hybrid DNA or with HAT hybrid protein. Control groupsof mice received only blank vector or alum adjuvant. Two weeks after thelast immunization, mice were challenged with 20 infective larvae ofBrugia malayi by placing them in a micropore chamber in the peritonealcavity of the immunized mice. After 48 hours, larval death was measuredto determine the success of vaccination.

FIG. 4 shows multivalent immunogenic composition-induced protectionagainst Brugia malayi infection in macaques. All animals (vaccinated andcontrol) were challenged with 130-180 L3s of Brugia malayi one monthafter the last immunization. In weeks 5, 10, 15 and 18 post-challenge,10 ml of blood was collected from each macaque between 18:00 and 22:00hours and screened for the presence of microfilariae using a modifiedKnott technique and analyzed by PCR for the Hha-1 repeats. Absence ofinfection in microfilaria (Mf)-negative animals was further confirmed bySXP-1 (B. malayi diagnostic antigen) ELISA. Results show thatrBmHAXT+AL019 (alum plus glucopyranosyl lipid adjuvant-stable emulsion)is a better immunogenic composition formulation than the otherformulations tested (n=10 per group). Chi-square test and Fisher's exacttest were used to compare the proportions across the groups.

FIG. 5 shows the results of an antibody-dependent cell-mediatedcytotoxicity (ADCC) assay. Approximately 10 Brugia malayi larvae wereincubated for 72 hours at 37° C. with 2×10⁵ peripheral blood mononuclearcells (PBMCs) and 50 μl of sera samples from each macaque. Larval deathin each well was monitored under a light microscope. Each data pointindicates the percent larval death using a serum sample from one animal.‘+’ indicates the average percent larvicidal activity for that group.n=10 macaques per group. *P≤0.005 compared with the AL019 (alum plusglucopyranosyl lipid adjuvant-stable emulsion) group. Statisticalanalysis was performed by a Kruskal-Wallis test followed by Bonferronicorrection for multiple tests. rBmHAXT, recombinant B. malayiHSP/ALT-2/TPX-2/TSPLEL.

FIG. 6 shows the results of an ADCC assay for killing of drug-sensitiveand drug-resistant Dirofilaria immitis in dogs. Approximately 8-10 D.immitis larvae were incubated for 96 hours at 37° C. with 0.5 millionPBMCs and 100 μl of sera samples from each dog. Larval death in eachwell was monitored under a light microscope. Each data point indicatesthe percent larval death using a serum sample from one animal. rBmHAXT,recombinant B. malayi HSP/ALT-2/TPX-2/TSPLEL.

DETAILED DESCRIPTION OF THE INVENTION

A multivalent immunogenic composition for filariasis has now beendeveloped. Combinations of antigens, such as Abundant Larval Transcript(ALT2), Tetraspanin (TSP), Small heat shock protein (HSP) 12.6, VespidVenom Allergen homologue-Like protein (VAL-1), Glutathione S-Transferase(GST), and Thioredoxin Peroxidase 2 (TPX-2), and fragments thereof, weretested in experimental animals (i.e., mouse, jirds, mastomys, macaque,and dogs) and shown to provide >80% protection against infection byfilarial nematodes such as Brugia malayi and Dirofilaria immitis.Accordingly, the present invention features protein-based and DNA-basedcompositions composed of filarial nematode antigens or nucleic acidsencoding the same and use of the immunogenic compositions to prevent orcontrol filariasis in humans and animals, in particular canids andfelids. In addition to vaccination, the present invention also providesassays and kits for detecting the presence of a filarial nematode.

For the purposes of the present invention, a multivalent or polyvalentimmunogenic composition refers to an immunogenic composition or vaccineprepared from several antigens. According to some embodiments, theantigen is a nucleic acid molecule, which is referred to herein as a“DNA-based” antigen. According to other embodiments, the antigen is aprotein or polypeptide, which is referred to herein as “protein-based”antigen. A multivalent immunogenic composition of the invention can becomposed of two, three, four, five, six or up to ten antigens or theirfragments in various permutation combinations. In particularembodiments, the multivalent immunogenic composition is composed of two,three or four antigens. In some embodiments, the multivalent immunogeniccomposition is composed of solely of protein antigens. In otherembodiments, the multivalent immunogenic composition is composed solelyof DNA-based antigens. In yet other embodiments, the multivalentimmunogenic composition is composed of a mixture of protein- andDNA-based antigens.

Antigens of the instant invention can be provided or expressed from asingle nucleic acid molecule containing, e.g., internal ribosome entrysites between the antigens. Moreover, the antigens of the multivalentimmunogenic composition of this invention can be covalently attached toform a hybrid or chimeric molecule or fusion protein, wherein theantigens are immediately adjacent to one another (e.g., an in-framefusion with or without a short spacer). Alternatively, antigens of theinstant invention can be provided as a mixture of individual antigens.Moreover, it is contemplated that the instant immunogenic compositioncan be composed of a hybrid molecule containing, e.g., two antigens, inadmixture with a third non-covalently attached antigen. By way ofillustration, a multivalent immunogenic composition of the invention canbe composed of a chimeric TSP-HSP protein in admixture with a nucleicacid molecule encoding ALT2.

In one embodiment, the antigens of the multivalent immunogeniccomposition are different proteins from one species of filarialnematode. As an example of this embodiment, the multivalent immunogeniccomposition is composed of ALT2, HSP, and TSP and/or TPX2 or GSTantigens isolated from one or more strains of B. malayi or D. immitis.In another embodiment, the antigens are the same, but from differentspecies of filarial nematodes. As an example of this embodiment, themultivalent immunogenic composition is composed of the ALT2 antigenisolated from W. bancrofti, B. malayi, B. timori, and D. immitis. In yeta further embodiment, the multivalent immunogenic composition iscomposed of a combination of different antigens from different speciesof filarial nematodes. By way of illustration, the multivalentimmunogenic composition can be composed of the ALT2 antigen isolatedfrom W. bancrofti, O. volvulus and L. loa and the HSP antigen isolatedfrom B. malayi and D. immitis.

For preparing multivalent DNA-based or multivalent recombinant DNA-basedimmunogenic composition, the DNA sequence of the gene of interest (alsoused interchangeably as DNA molecule) need not contain the full lengthof DNA encoding the corresponding protein. Likewise, when preparingfusion protein-based or multivalent recombinant protein immunogeniccompositions, the protein sequence need not contain the full-lengthprotein. In most cases, a fragment of the protein or gene which encodesan epitope region is sufficient for immunization. The DNA/proteinsequence of an epitope region can be found by sequencing thecorresponding part of the gene from various strains or species andcomparing them. The major antigenic determinants are likely to be thoseshowing the greatest heterology. Also, these regions are likely to lieaccessibly in the conformational structure of the proteins. One or moresuch fragments of proteins or genes encoding the antigenic determinantscan be prepared by chemical synthesis or by recombinant DNA technology.These fragments of proteins or genes, if desired, can be linked togetheror linked to other proteins or DNA molecules, respectively.

As described herein, the ALT2, TSP, VAL-1, GST and HSP antigens wereidentified as providing protection against infection by filaria larvae.Accordingly, in particular embodiments, the instant immunogeniccomposition includes the ALT2, TSP, VAL-1, TPX2, GST and/or HSP proteinantigens and/or nucleic acid molecules encoding the ALT2, TSP, VAL-1,TPX2, GST and/or HSP protein, or fragments thereof. Protein and nucleicacid sequences for these antigens are available under the GENBANKaccession numbers and/or sequences listed in Table 1.

TABLE 1 SEQ ID SEQ ID Antigen Source Protein NO: Nucleic Acid NO: ALT2B. malayi P90708 37 BMU84723 38 XP_001896203 39 XM_001896168 40 W.bancrofti AAC35355 41 AF084553 42 L. loa XP_003151340 43 XM_003151292 44D. immitis AAC47031 93 — 92 TSP B. malayi ABN55911 45 EF397425 46 L. loaXP_003136177 47 XM_003136129 48 HSP B. malayi AAU04396 49 AY692227 50 O.volvulus CAA48633 51 X68669 52 L. loa XP_003139338 53 XM_003139290 54 D.immitis QHA79233 91 — 90 VAL-1 B. malayi AAB97283 55 AF042088 56 W.bancrofti AAD16985 57 AF109794 58 O. volvulus AAB69625 59 AF020586 60 L.loa XP_003146897 61 XM_003146849 62 TPX2 B. malayi Q17172 71 U47100 72D. immitis AAC38831 95 — 94 GST W. bancrofti AA045827 85 AY195867 86 D.immitis P46426 103 — 102 B. malayi XP_001898233 120 — —

In addition, the nucleotide sequence encoding O. volvulus TSP can befound under GENBANK Accession No. JN861043. The protein antigens andnucleic acid molecules of the invention can be used as full lengthmolecules or less than full length molecules. In this respect, thepresent invention further includes the use of fragments of theabove-referenced protein antigens and nucleic acid molecules. Fragmentsare defined herein as 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200amino acid residue portions of full-length protein antigens (e.g., thoselisted in Table 1) or 60, 90, 120, 150, 180, 210, 240, 270, 300, 350, or600 nucleotide portion of full-length nucleic acid molecules (e.g.,those listed in Table 1). Exemplary protein fragments include the largeextracellular loop (LEL) domain of TSP (see, e.g., the LEL domain of B.malayi TSP of SEQ ID NO:63 or SEQ ID NO:77) and N-terminal deletion ofHSP 12.6 (cHSP; see, e.g., the B. malayi HSP fragment of SEQ ID NO:64),as well as the nucleic acid molecules encoding the same (see, SEQ IDNO:65 and SEQ ID NO:66, respectively). An exemplary fusion proteincontaining ALT2, HSP and TSP protein sequences is set forth in SEQ IDNO:70. An exemplary fusion protein containing ALT2, HSP and TPX2 proteinsequences is set forth in SEQ ID NO:73 and SEQ ID NO:97. An exemplaryfusion protein containing ALT2, HSP, TSP and TPX2 protein sequences isset forth in SEQ ID NO:74.

In particular embodiments, the protein or protein fragments of thisinvention have one or more antigenic sequences for eliciting an immuneresponse in an animal. In certain embodiments, the ALT2 protein of theinvention is a B. malayi ALT2 protein or fragment comprising orconsisting of the sequence VSESDEEFDDSAADDTDDSEAGGGSEGGDEYVT (SEQ IDNO:78) and/or EFVETDGKKKECSSHEACYDQREPQ (SEQ ID NO:79) or D. immitisALT2 protein or fragment comprising or consisting of the sequenceASESQEETVSFEESDEDYEDDSE (SEQ ID NO:98) and/or FVESDGKMKHCKTHEACYDQREPQ(SEQ ID NO:99), which, based upon the Bepipred Linear Epitope Predictionmethod (Larsen, et al. (2006) Immunome Res. 2:2), are predicted B-cellepitopes. In other embodiments, the HSP protein of the invention is a B.malayi HSP protein or fragment comprising or consisting of the sequenceWSAEQWDWPLQH (SEQ ID NO:80) and/or KLPSDVDTKTL (SEQ ID NO:81) or D.immitis HSP protein or fragment comprising or consisting of the sequenceNWSADQWDWPLQHNDDVVKVTNTNDK (SEQ ID NO:100) and/or KLPSDVDTKTL (SEQ IDNO:81), which are predicted B-cell epitopes. In further embodiments, theTSP protein of the invention is a B. malayi TSP protein or fragmentcomprising or consisting of the sequence KTGESEDEMQ (SEQ ID NO:82),which is a predicted B-cell epitope. In yet a further embodiment, theTPX2 protein of the invention is a B. malayi TPX2 protein or fragmentcomprising or consisting of the sequence FIGQPAPNFKT (SEQ ID NO:83)and/or GEVCPANWHPGSETIKPGVKESKA (SEQ ID NO:84) or D. immitis TPX2protein or fragment comprising or consisting of the sequence FIGQPAPNFKT(SEQ ID NO:83) and/or GEVCPANWQPGSEAIKPGVKESKA (SEQ ID NO:101), whichare predicted B-cell epitopes.

In certain embodiments, ALT2 protein fragments of this inventioncomprise or consist of the amino acid sequences X₁X₂ESDEX₃X₄X₅DX₆ (SEQID NO:121), wherein independently X₁ is V or F, X₂ is S or E, X₃ is E orD, X₄ is F or Y, X₅ is D or E, and X₆ is S or D; orFVEX₁DGKX₂KX₃CX₄X₅HEACYDQREPQ (SEQ ID NO:122), wherein independently X₁is S or T; X₂ is M or K; X₃ is E or H, X₄ is S or K, and X₅ is S or T.In other embodiments, HSP protein fragments of this invention compriseor consist of the amino acid sequences WSAX₁QWDWPLQH (SEQ ID NO:123),wherein independently X₁ is Glu or Asp; or KLPSDVDTKTL (SEQ ID NO:81).In further embodiments, TPX2 protein fragments of this inventioncomprise or consist of the amino acid sequences FIGQPAPNFKT (SEQ IDNO:83); or GEVCPANWX₁PGSEX₂IKPGVKESKA (SEQ ID NO:124), whereinindependently X₁ is H or Q, and X₂ is T or A.

With respect to certain embodiments of the invention, the multivalentimmunogenic composition of the invention includes other known antigensfrom filarial nematodes. Examples of other suitable antigens include,but are not limited to, glutathione peroxidase (see Cookson, et al.(1992) Proc. Natl. Acad. Sci. USA 89:5837-5841; Maizels, et al. (1983)Parasitology 87:249-263; Maizels, et al. (1983) Clin. Exp. Immunol.51:269-277); recombinant antigen (BmR1; see Noordin, et al. (2004)Filaria J. 3:10); class II aminoacyl-tRNA synthetase (see Kron, et al.(1995) FEBS Lett. 374:122-4); heat shock cognate 70 (hsc70) protein (seeSelkirk, et al. (1989) J. Immunol. 143:299-308); paramyosin (see Li, etal. (1991) Mol. Biochem. Parasitol. 49:315-23); tropomyosin (Hartmann,et al. (2006) Vaccine 24(17):3581-90); chitinase (Adam, et al. (1996) J.Biol. Chem. 271(3):1441-7); Abundant Larval Transcript (ALT)-1 (Gregory,et al. (2000) Infect. Immun. 68(7):4174-9); immunodominant hypodermalantigen SPX1 (Bradley, et al. (1993) Exp. Parasitol. 77(4):414-424). Insome embodiments, the antigen is obtained from a filarial nematodeselected from the group of W. bancrofti, B. malayi, O. volvulus, L. loa,D. immitis and B. timori. In certain embodiments, the antigen is B.malayi or Dirofilaria tropomyosin having an amino acid sequence as setforth in SEQ ID NO:104 and SEQ ID NO:105, respectively, or a fragmentthereof; B. malayi or Dirofilaria chitinase having an amino acidsequence as set forth in SEQ ID NO:106 and SEQ ID NO:107, respectively,or a fragment thereof; B. malayi or Dirofilaria ALT-1 having an aminoacid sequence as set forth in SEQ ID NO:108 and SEQ ID NO:109,respectively, or a fragment thereof; B. malayi or Dirofilaria SPX1having an amino acid sequence as set forth in SEQ ID NO:110 and SEQ IDNO:111, respectively, or a fragment thereof; B. malayi or D. immitisvenom allergen antigen 5-like protein having an amino acid sequence asset forth in SEQ ID NO:112 and SEQ ID NO:113, respectively, or afragment thereof; B. malayi or D. immitis Macrophage migrationInhibitory Factor (MIF)-1 protein having an amino acid sequence as setforth in SEQ ID NO:114 and SEQ ID NO:115, respectively, or a fragmentthereof; B. malayi or Dirofilaria MIF-2 protein having an amino acidsequence as set forth in SEQ ID NO:116 and SEQ ID NO:117, respectively,or a fragment thereof; or B. malayi or Dirofilaria cystatin proteinhaving an amino acid sequence as set forth in SEQ ID NO:118 and SEQ IDNO:119, respectively, or a fragment thereof.

According to the present invention, the antigens of the fusion proteinand immunogenic composition are isolated from a filarial nematode. Inthis respect, an isolated nucleic acid molecule or protein is a nucleicacid molecule or protein that has been removed from its natural milieu(i.e., that has been subjected to human manipulation). As such,“isolated” does not reflect the extent to which the nucleic acidmolecule or protein has been purified. In particular embodiments, theantigens are purified (e.g., purified to greater than 95% homogeneity).An isolated and optionally purified nucleic acid molecule or protein ofthe present invention can be obtained from its natural source orproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification or cloning) or chemical synthesis. Isolatednucleic acid molecules and proteins can also include, for example,natural allelic variants or isomers that induce an immune response inthe host.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into a vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that are nucleic acid sequencesthat are not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either prokaryotic or eukaryotic, and typically is avirus or a plasmid. Recombinant vectors can be used in the cloning,sequencing, and/or otherwise manipulating the nucleic acid molecules ofthe present invention.

The present invention also includes an expression vector, which includesa nucleic acid molecule of the present invention in a recombinant vectorthat is capable of expressing the nucleic acid molecule when transformedinto a host cell. Preferably, the expression vector is also capable ofreplicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, parasite, insect,other animal, and plant cells. Preferred expression vectors of thepresent invention can direct gene expression in bacterial, yeast,helminth or other parasite, insect and mammalian cells.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, helminth or otherendoparasite, or insect and mammalian cells, such as, but not limitedto, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda(such as lambda P_(L) and lambda P_(R) and fusions that include suchpromoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophageSP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichiaalcohol oxidase, alphavirus subgenomic promoter, antibiotic resistancegene, baculovirus, Heliothis zea insect virus, vaccinia virus,herpesvirus, raccoon poxvirus, other poxvirus, adenovirus,cytomegalovirus (such as immediate early promoter), simian virus 40,retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus,heat shock, phosphate and nitrate transcription control sequences aswell as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins). Transcription control sequences of thepresent invention can also include naturally occurring transcriptioncontrol sequences naturally associated with parasitic helminths, such asB. malayi or D. immitis transcription control sequences.

Recombinant molecules of the present invention may also contain (a)secretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed protein of the present invention to be secreted fromthe cell that produces the protein and/or (b) fusion sequences whichlead to the expression of nucleic acid molecules of the presentinvention as fusion proteins. Examples of suitable signal segmentsinclude any signal segment capable of directing the secretion of aprotein of the present invention. Preferred signal segments include, butare not limited to, tissue plasminogen activator (t-PA), interferon,interleukin, growth hormone, histocompatibility and viral envelopeglycoprotein signal segments. In addition, a nucleic acid molecule ofthe present invention can be joined to a fusion segment that directs theencoded protein to the proteosome, such as a ubiquitin fusion segment.Eukaryotic recombinant molecules may also include intervening and/oruntranslated sequences surrounding and/or within the nucleic acidsequences of nucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant hostcell harboring one or more recombinant molecules of the presentinvention. Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent immunogeniccompositions). Host cells of the present invention either can beendogenously (i.e., naturally) capable of producing proteins of thepresent invention or can be capable of producing such proteins afterbeing transformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), parasite (including helminth,protozoa and ectoparasite), other insect, other animal and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, helminth,insect and mammalian cells. More preferred host cells includeSalmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera,Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells(Madin-Darby canine kidney cell line), CRFK cells (Crandell felinekidney cell line), CV-1 cells (African monkey kidney cell line used, forexample, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Verocells. Particularly preferred host cells are Escherichia coli, includingE. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium;Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFKcells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mousemyoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriatemammalian cell hosts include other kidney cell lines, other fibroblastcell lines (e.g., human, murine or chicken embryo fibroblast celllines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3cells, LMTK³¹ cells and/or HeLa cells. In one embodiment, the proteinsmay be expressed as heterologous proteins in myeloma cell linesemploying immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention and one or moretranscription control sequences, examples of which are disclosed herein.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein. Moreover, while non-codon-optimized sequences may be used toexpress fusion proteins in host cells such as E. coli (see Table 1), inembodiments pertaining to DNA vaccines, the nucleic acid molecule may becodon-optimized to facilitate expression in mammalian cells. In thisrespect, codon-optimized sequences for BmALT2, N-terminal deleted HSP12.6 (cHSP) of B. malayi, and LEL domain of B. malayi Tetraspanin areset forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69, respectively.Moreover, to facilitate expression of one or more of the recombinantproteins in a recombinant host cell, the protein sequence can bemanipulated. By way of illustration, the insertion of a glycine residueafter the N-terminal methionine residue of the B. malayi ALT2 proteinwas found to improve expression of this protein in E. coli.

Isolated protein-based antigens of the present invention can be producedin a variety of ways, including production and recovery of naturalproteins, production and recovery of recombinant proteins, and chemicalsynthesis of the proteins. In one embodiment, an isolated protein of thepresent invention is produced by culturing a cell capable of expressingthe protein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective, mediumrefers to any medium in which a cell is cultured to produce a protein ofthe present invention. Such medium typically includes an aqueous mediumhaving assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. Cells of the present invention can be cultured in conventionalfermentation bioreactors, shake flasks, test tubes, microtiter dishes,and petri plates. Culturing can be carried out at a temperature, pH andoxygen content appropriate for a recombinant cell. Such culturingconditions are within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

Recovery of proteins of invention can include collecting the wholefermentation medium containing the protein and need not imply additionalsteps of separation or purification. Proteins of the present inventioncan be purified using a variety of standard protein purificationtechniques, such as, but not limited to, affinity chromatography, ionexchange chromatography, filtration, electrophoresis, hydrophobicinteraction chromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization. Proteins of the present invention arepreferably retrieved in substantially pure form thereby allowing for theeffective use of the protein as a therapeutic composition. A therapeuticcomposition for animals, for example, should exhibit no substantialtoxicity and preferably should be capable of stimulating the productionof antibodies in a treated animal.

One embodiment of the present invention is an immunogenic composition orvaccine that, when administered to an animal in an effective manner, iscapable of protecting that animal from filariasis or dirofilariasiscaused by a filarial nematode such as a Dirofilaria nematode. In someembodiments, the invention provides a method for treating or protectingan animal from a disease caused by a filarial nematode. In otherembodiments, the invention provides a method for treating or protectingan animal, e.g., a dog or cat, from dirofilariasis (heartworm disease).Immunogenic compositions include protective molecules such as anisolated antigenic protein of the present invention, an isolated nucleicacid molecule of the present invention, and hybrids and mixturesthereof. As used herein, the multivalent immunogenic composition of theinvention induces a protective immune response when administered in aneffective manner to an animal such as a human, cat or dog therebytreating, ameliorating, and/or preventing disease caused by a filarialor dirofilarial nematode including, but not limited to, W. bancrofti, B.malayi, O. volvulus, L. loa, D. immitis, Mansonella streptocerca,Dracunculus medinensis, M. perstans, M. ozzardi, and/or B. timori.Immunogenic composition of the present invention can be administered toany animal susceptible to such therapy, preferably to mammals, and morepreferably to humans, pets such as dogs and cats, and economic foodanimals and/or zoo animals.

In one embodiment, a multivalent immunogenic composition of the presentinvention when administered to the host can develop antibodies that cankill the parasites in the vector in which the filarial nematodedevelops, such as in a mosquito when they feed the host.

In order to protect an animal from disease caused by a filarialnematode, an immunogenic composition of the present invention isadministered to the animal in an effective manner such that thecomposition is capable of protecting that animal from a disease causedby the filarial nematode. Compositions of the present invention can beadministered to animals prior to infection in order to prevent infection(i.e., as a preventative vaccine) and/or can be administered to animalsafter infection in order to treat disease caused by the filarialnematode (i.e., as a therapeutic vaccine).

Compositions of the present invention can be formulated in an excipientthat the animal to be treated can tolerate. Examples of such excipientsinclude water, saline, Ringer's solution, dextrose solution, Hank'ssolution, and other aqueous physiologically balanced salt solutions.Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, ortriglycerides may also be used. Other useful formulations includesuspensions containing viscosity enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the immunogenic compositioncan include an adjuvant. An “adjuvant,” as defined herein, is asubstance that serves to enhance the immunogenicity of an immunogeniccomposition of the invention. An immune adjuvant may enhance an immuneresponse to an antigen that is weakly immunogenic when administeredalone, e.g., inducing no or weak antibody titers or cell-mediated immuneresponse, increase antibody titers to the antigen, and/or lowers thedose of the antigen effective to achieve an immune response in theindividual. Thus, adjuvants are often given to boost the immune responseand are well known to the skilled artisan.

Suitable adjuvants to enhance effectiveness of the immunogeniccomposition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) calcium-based salts;

(3) silica;

(4) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example,

-   -   (a) MF59 (WO 90/14837), containing 5% squalene, 0.5% polysorbate        80, and 0.5% sorbitan trioleate (optionally containing various        amounts of muramyl tripeptide phosphatidylethanolamine)        formulated into submicron particles using a microfluidizer such        as Model 110Y microfluidizer (Microfluidics, Newton, Mass.),    -   (b) SAF, containing 10% squalene, 0.4% polysorbate 80, 5%        pluronic-blocked polymer L121, and thr-MDP either microfluidized        into a submicron emulsion or vortexed to generate a larger        particle size emulsion,    -   (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.)        containing 2% squalene, 0.2% polysorbate 80, and one or more        bacterial cell wall components from the group consisting of        3-O-deacylated monophosphorylipid A (MPL®) described in U.S.        Pat. No. 4,912,094, trehalose dimycolate (TDM), and cell wall        skeleton (CWS), preferably MPL+CWS (Detox™); and    -   (d) a Montanide ISA;

(5) saponin adjuvants, such as those sold under the tradenames QUIL-A®or QS-21 STIMULON® (Antigenics, Framingham, Mass.) (see, e.g., U.S. Pat.No. 5,057,540), may be used or particles generated therefrom such asISCOM (immunostimulating complexes formed by the combination ofcholesterol, saponin, phospholipid, and amphipathic proteins) andIscomatrix™ (having essentially the same structure as an ISCOM butwithout the protein);

(6) bacterial components (e.g., endotoxins, in particular superantigens,exotoxins and cell wall components) and lipopolysaccharides, syntheticlipid A analogs such as aminoalkyl glucosamine phosphate compounds(AGP), or derivatives or analogs thereof, which are available fromCorixa, and described in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxy-tetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion;

(7) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646);

(8) cytokines and chemokines (e.g., granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF), colonystimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2),IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18,interferon gamma, interferon gamma inducing factor I (IGIF),transforming growth factor beta, RANTES (regulated upon activation,normal T-cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), tumor necrosisfactor (TNF), costimulatory molecules B7-1 and B7-2, and Leishmaniaelongation initiating factor (LEIF));

(9) complement, such as a trimer of complement component C3d;

(10) toll-like receptor agonists, e.g., TLR4 agonists such asglucopyranosyl lipid adjuvant (GLA);

(11) serum proteins, e.g., transferrin;

(12) viral coat proteins, e.g., rotavirus capsid VP6 protein; and

(13) block copolymer adjuvants, e.g., Hunter's TITERMAX® adjuvant(VAXCEL, Inc. Norcross, Ga.).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Protein adjuvants of the present invention can be delivered in the formof the protein themselves or of nucleic acid molecules encoding suchproteins using the techniques described herein.

In certain embodiments, the adjuvant includes an aluminum salt. Thealuminum salt adjuvant may be an alum-precipitated vaccine or analum-adsorbed vaccine. Aluminum-salt adjuvants are well-known in the artand are described, for example, in Harlow & Lane ((1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory) and Nicklas ((1992)Res. Immunol. 143:489-493). The aluminum salt includes, but is notlimited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH),aluminum hydrate, aluminum trihydrate, aluminum (III) hydroxide,aluminum hydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA),amorphous alumina, trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g., aluminumhydroxide gel sold under the tradename Alhydrogel®) is used to adsorbproteins in a ratio of 50-200 μg protein/mg aluminum hydroxide.Adsorption of protein is dependent, in another embodiment, on the pI(Isoelectric pH) of the protein and the pH of the medium. A protein witha lower pI adsorbs to the positively charged aluminum ion more stronglythan a protein with a higher pI. Aluminum salts may establish a depot ofantigen that is released slowly over a period of 2-3 weeks, be involvedin nonspecific activation of macrophages and complement activation,and/or stimulate innate immune mechanism (possibly through stimulationof uric acid). See, e.g., Lambrecht, et al. (2009) Curr. Opin. Immunol.21:23.

In some embodiments, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS-21); or alum in combinationwith GLA (AL019).

The multivalent immunogenic composition of the invention can beformulated as single dose vials, multi-dose vials or as pre-filled glassor plastic syringes.

In one embodiment, multivalent immunogenic compositions of the presentinvention are administered orally, and are thus formulated in a formsuitable for oral administration, i.e., as a solid or a liquidpreparation. Solid oral formulations include tablets, capsules, pills,granules, pellets and the like. Liquid oral formulations includesolutions, suspensions, dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor non-aqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However, it is often preferred that a composition for infusion orinjection is essentially isotonic, when it is administrated. Hence,storage of the composition may preferably be isotonic or hypertonic. Ifthe composition is hypertonic for storage, it may be diluted to becomean isotonic solution prior to administration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂, KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thecomposition is meant for infusion or injection, it is often desirablethat the composition includes a buffer, which is capable of buffering asolution to a pH in the range of 4 to 10, such as 5 to 9, for example 6to 8.

The buffer may, for example, be selected from Tris, acetate, glutamate,lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate,histidine, glycine, succinate and triethanolamine buffer. The buffer maybe selected from USP compatible buffers for parenteral use, inparticular, when the formulation is for parenteral use. For example thebuffer may be selected from the group of monobasic acids such as acetic,benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic,adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,polybasic acids such as citric and phosphoric; and bases such asammonia, diethanolamine, glycine, triethanolamine, and Tris.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations of the invention may also contain a surfactant.Preferred surfactants include, but are not limited to, thepolyoxyethylene sorbitan esters surfactants, especially PS-20 and PS-80;copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butyleneoxide (BO), sold under the tradename DOWFAX™, such as linear EO/PO blockcopolymers; octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters, such as sorbitan trioleate and sorbitan monolaurate. A preferredsurfactant for including in the emulsion is PS-80.

Mixtures of surfactants can be used. A combination of a polyoxyethylenesorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) andan octoxynol such as t-octylphenoxypolyethoxyethanol is also suitable.Another useful combination comprises laureth 9 plus a polyoxyethylenesorbitan ester and/or an octoxynol.

Poloxamer may also be used in the compositions of the invention. Apoloxamer is a nonionic triblock copolymer composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).Poloxamers are also known by the tradename Pluronic®. Because thelengths of the polymer blocks can be customized, many differentpoloxamers exist that have slightly different properties. For thegeneric term “poloxamer”, these copolymers are commonly named with theletter “P” (for poloxamer) followed by three digits, the first twodigits×100 give the approximate molecular mass of the polyoxypropylenecore, and the last digit×10 gives the percentage polyoxyethylene content(e.g., P407=Poloxamer with a polyoxypropylene molecular mass of 4,000g/mol and a 70% polyoxyethylene content). For the Pluronic® tradename,coding of these copolymers starts with a letter to define its physicalform at room temperature (L=liquid, P=paste, F=flake (solid)) followedby two or three digits. The first digit (two digits in a three-digitnumber) in the numerical designation, multiplied by 300, indicates theapproximate molecular weight of the hydrophobe; and the last digit×10gives the percentage polyoxyethylene content (e.g., L61 is a Pluronic®with a polyoxypropylene molecular mass of 1,800 g/mol and a 10%polyoxyethylene content). See U.S. Pat. No. 3,740,421.

Preferably, the poloxamer generally has a molecular weight in the rangefrom 1100 to 17,400 Da, from 7,500 to 15,000 Da, or from 7,500 to 10,000Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407.The final concentration of the poloxamer in the formulations is from0.001% to 5% weight/volume, or 0.025% to 1% weight/volume. In certainaspects, the polyol is propylene glycol and is at final concentrationfrom 1% to 20% weight/volume. In certain aspects, the polyol ispolyethylene glycol 400 and is at final concentration from 1% to 20%weight/volume.

Suitable polyols for the formulations of the invention are polymericpolyols, particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from about 425 to about 2700. Polyethylene glycol andPolyethylene glycol monomethyl ether is also available in a range ofmolecular weights ranging from about 200 to about 35000 including butnot limited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferredpolyethylene glycol is polyethylene glycol 400. The final concentrationof the polyol in the formulations of the invention may be 1% to 20%weight/volume or 6% to 20% weight/volume.

The formulation may also contain a pH-buffered saline solution. Thebuffer may, for example, be selected from the group consisting of Tris,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspects of the invention, thebuffer is selected from the group of phosphate, succinate, histidine,MES, MOPS, HEPES, acetate or citrate. The buffer may furthermore, forexample, be selected from USP compatible buffers for parenteral use, inparticular, when the pharmaceutical formulation is for parenteral use.The concentrations of buffer will range from 1 mM to 100 mM. Theconcentrations of buffer will range from 10 mM to 80 mM. Theconcentrations of buffer will range from 1 mM to 50 mM or 5 mM to 50 mM.

While the saline solution (i.e., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂, KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but are not limited to 25 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 20 mM to 170 mM.

In some aspects, the composition of the invention is administered to asubject by one or more methods known to a person skilled in the art,such as parenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly. In one embodiment, acomposition of the present invention is administered via epidermalinjection, intramuscular injection, intravenous, intra-arterial,subcutaneous injection, or intra-respiratory mucosal injection of aliquid preparation. Liquid formulations for injection include solutionsand the like.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation includes a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation is capable of releasing animmunogenic composition of the present invention into the blood of thetreated animal at a constant rate sufficient to attain therapeutic doselevels of the composition to protect an animal from disease caused by afilarial nematode. For example, the immunogenic composition can beadministered using intravenous infusion, a transdermal patch, liposomes,or other modes of administration. In another embodiment, polymericmaterials are used, e.g., in microspheres in or an implant. Theimmunogenic composition is preferably released over a period of timeranging from about 1 to about 12 months. A controlled releaseformulation of the present invention is capable of effecting a treatmentpreferably for at least about 1 month, more preferably for at leastabout 3 months, even more preferably for at least about 6 months, evenmore preferably for at least about 9 months, and even more preferablyfor at least about 12 months.

Immunogenic compositions or vaccines of the present invention can beadministered to animals prior to infection in order to prevent infectionand/or can be administered to animals after infection in order to treatdisease caused by a filarial nematode. For example, proteins, nucleicacids and mixtures thereof can be used as immunotherapeutic agents.Acceptable protocols to administer compositions in an effective mannerinclude individual dose size, number of doses, frequency of doseadministration, and mode of administration. Determination of suchprotocols can be accomplished by those skilled in the art. A suitablesingle dose is a dose that is capable of protecting an animal fromdisease when administered one or more times over a suitable time period.For example, a preferred single dose of a protein-based vaccine is fromabout 1 microgram (pg) to about 10 milligrams (mg) of protein-basedvaccine per kilogram body weight of the animal. Booster vaccinations canbe administered from about 2 weeks to several years after the originaladministration. Booster administrations preferably are administered whenthe immune response of the animal becomes insufficient to protect theanimal from disease. A preferred administration schedule is one in whichfrom about 10 μg to about 1 mg of the vaccine per kg body weight of theanimal is administered from about one to about two times over a timeperiod of from about 2 weeks to about 12 months. Modes of administrationcan include, but are not limited to, subcutaneous, intradermal,intravenous, intranasal, oral, transdermal and intramuscular routes.

Wherein the immunogenic composition includes a nucleic acid molecule,the immunogenic composition can be administered to an animal in afashion to enable expression of that nucleic acid molecule into aprotective protein in the animal. Nucleic acid molecules can bedelivered to an animal in a variety of methods including, but notlimited to, administering a naked (i.e., not packaged in a viral coat orcellular membrane) nucleic acid as a genetic vaccine (e.g., as naked DNAmolecules, such as is taught, for example in Wolff, et al. (1990)Science 247:1465-1468); or administering a nucleic acid moleculepackaged as a recombinant virus vaccine or as a recombinant cell vaccine(i.e., the nucleic acid molecule is delivered by a viral or cellularvehicle).

A genetic (i.e., naked nucleic acid) vaccine of the present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplification, competent. A genetic vaccineof the present invention can include one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. Preferred genetic vaccines include at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses (such as sindbis or Semliki forest virus), species-specificherpesviruses and poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of a “strong” polyadenylation signalis also preferred.

Genetic vaccines of the present invention can be administered in avariety of ways, including intramuscular, subcutaneous, intradermal,transdermal, intranasal and oral routes of administration. Moreover, itis contemplated that the vaccine can be delivered by gene gun, skinpatch, electroporation, or nano-based delivery. In this respect,DNA-based and protein-based vaccines can be administered at the sametime. A preferred single dose of a genetic vaccine ranges from about 1nanogram (ng) to about 600 μg, depending on the route of administrationand/or method of delivery, as can be determined by those skilled in theart. Suitable delivery methods include, for example, by injection, asdrops, aerosolized and/or topically. Genetic vaccines of the presentinvention can be contained in an aqueous excipient (e.g.,phosphate-buffered saline) alone or in a carrier (e.g., lipid-basedvehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient and/or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picornaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. Examples of methods to produce and use alphavirusrecombinant virus vaccines are disclosed in PCT Publication No. WO94/17813.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein that is capable of protecting theanimal from filariasis caused by filarial nematodes. By way ofillustration, a single dose of a recombinant virus vaccine of thepresent invention can be from about 1×10⁴ to about 1×10⁸ virus plaqueforming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal andoral as routes of administration.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express a protein of the presentinvention. Preferred recombinant cells for this embodiment includeSalmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast,(including Saccharomyces cerevisiae and Pichia pastoris), BHK, CV-1,myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells.Recombinant cell vaccines of the present invention can be administeredin a variety of ways but have the advantage that they can beadministered orally, preferably at doses ranging from about 10⁸ to about10¹² cells per kilogram body weight. Administration protocols aresimilar to those described herein for protein-based vaccines.Recombinant cell vaccines can include whole cells, cells stripped ofcell walls or cell lysates.

In some embodiments of the composition of the invention, all of theantigens are present in the composition in the same amount. In furtherembodiments, the antigens are present in the composition in differentamounts (i.e., at least one antigen is present in an amount that isdifferent than one or more of the other antigens of the composition).

Optimal amounts of components for a particular immunogenic compositioncan be ascertained by standard studies involving observation ofappropriate immune responses in subjects. For example, in anotherembodiment, the dosage for human vaccination is determined byextrapolation from animal studies to human data. In another embodiment,the dosage is determined empirically.

As is known in the art, there are three groups of filarial nematodes,classified according to the niche within the body that they occupy:lymphatic filariasis, subcutaneous filariasis, and serous cavityfilariasis. Lymphatic filariasis is caused by the worms W. bancrofti, B.malayi and B. timori. These worms occupy the lymphatic system, includingthe lymph nodes, and cause fever, lymphadenitis (swelling of the lymphnodes), lymphangitis (inflammation of the lymphatic vessels in responseto infection), and lymphedema (elephantiasis). Subcutaneous filariasismay be caused by Loa loa (the African eye worm), Mansonella stretocerca,O. volvulus, Dracunculus medinensis, or Dirofilaria immitis. Many ofthese worms occupy the subcutaneous layer of the skin, in the fat layer,and present with skin rashes, urticarial papules, and arthritis, as wellas hyper- and hypopigmentation macules. Onchocerca volvulus manifestsitself in the eyes, causing “river blindness.” Adult Dirofilaria immitisreside in pulmonary arteries and are the causal agent of heartwormdisease. Serous cavity filariasis is caused by the worms M. perstans andM. ozzardi, which occupy the serous cavity of the abdomen. Serous cavityfilariasis presents with symptoms similar to subcutaneous filariasis, inaddition to abdominal pain, because these worms are also deep tissuedwellers.

Dogs infected with Brugia malayi develop clinical lymphedema, scrotalenlargement, conjunctivitis and lymphagitis similar to the humanlymphatic filariasis; however, the pathology is not as severe as in thehuman. Since dogs carry the infection in the nature, humans can get theBrugia malayi infections from dogs. Thus, zoonotic infections are commonin the endemic areas, where dogs and cats carry the infection in thenature and they transmit the infection to the humans. Dogs and cats canalso be infected with Brugia malayi under laboratory conditions. Thus,an immunogenic composition developed against lymphatic filariasis indogs are also important in blocking transmission of the disease in thehuman.

The efficacy of a multivalent immunogenic composition of the presentinvention to protect an animal from filariasis or dirofilariasis causedby filarial nematodes can be tested in a variety of ways including, butnot limited to, detection of protective antibodies (using, for example,proteins of the present invention), detection of cellular immunitywithin the treated animal, and/or challenge of the treated animal withthe a filarial nematode to determine whether the treated animal isresistant to disease and fails to exhibit one or more signs of disease.Challenge studies can include implantation of chambers includingfilarial nematode larvae into the treated animal and/or directadministration of larvae to the treated animal. In one embodiment,therapeutic compositions can be tested in animal models such as mice,jirds (Meriones unguiculatus), mastomys (e.g., Mastomys natalensis)and/or dogs. Such techniques are known to those skilled in the art.

To detect the presence/amount of anti-filarial nematode antibodies,e.g., protective or neutralizing antibodies resulting from thevaccination of an animal, this invention also provides a method and kitfor efficacy evaluation, as well as for detecting prior exposure tofilarial proteins and/or infection with a filarial nematode. Inaccordance with such a method, one or more antigenic proteins/epitopesis contacted with a biological sample from an animal and binding betweenthe antigenic proteins/epitopes and antibodies in the biological sampleis quantitatively or qualitatively determined as described herein,wherein the presence and/or amount of antibodies to the antigenicproteins/epitopes is indicative of vaccine efficacy, as well as priorexposure to filarial proteins or an existing infection with a filarialnematode. In certain embodiments, the method and kit use an array-basedformat in which serial dilutions of one or more antigens or epitopes areprinted. In some embodiments, the one or more of the filarial nematodeproteins are present on one or more solid surfaces or particles. Inother embodiments, the one or more of the filarial nematode proteins arein an array so that the presence of multiple antibodies can be assessedin a single assay due to the multiplexing capability of an array-basedapproach. In this respect, the array can contain one or more of ALT2,TSP, VAL-1, TPX2, GST or HSP protein or an epitope thereof. In otherembodiments, the array at least contains each of the proteins used inthe multivalent immunogenic composition. For example, to assay forprotective or neutralizing antibodies against a multivalent immunogeniccomposition containing HSP, ALT2 and TSP, the array would contain HSP,ALT2 and TSP, or a fusion protein thereof.

For testing for the presence of a filarial nematode, this invention alsoprovides a method and kit for detecting a filarial nematode. The assaymethod generally includes the steps of contacting, in vitro, abiological sample with one or more binding agents against filarialnematode proteins selected from the group of ALT2, TSP, VAL-1, TPX2, GSTand HSP or fragments thereof. The bound binding agents are thendetected. The bound binding agents can be detected using automateddetection of binding such as an image reader of an ELISA assay, and if abound binding agent is detected, the data indicating that a boundbinding agent has been detected can be transferred, e.g., to a computerdisplay or on a paper print out. Detection of a filarial nematodeprotein indicates that the sample or subject from which the sample wasobtained has filariasis. Therefore, detection allows selection oftreatment options for the subject. Thus, in one embodiment, if one ormore of ALT2, TSP, VAL-1, TPX2, GST and HSP is detected, the patientwill be given a treatment suitable for filariasis, including but notlimited to treatment with diethylcarbamazine, mebendazole, flubendazole,albendazole, ivermectin or a combination thereof.

A biological sample is any material to be tested for the presence oramount of a protein of interest (e.g., an antibody or antigen/epitope).The sample can be a fluid sample, preferably a liquid sample. Examplesof liquid samples that may be tested in accordance with this inventioninclude bodily fluids including blood, serum, plasma, saliva, urine,ocular fluid, semen, and spinal fluid. Viscous liquid, semi-solid, orsolid specimens (e.g., human tissue, or mosquito or fly tissue) may beused to create liquid solutions, eluates, suspensions, or extracts thatcan be samples. In some embodiments, the biological sample is undiluted.In other embodiments, the sample is diluted or concentrated depending onthe detection application.

In certain embodiments, one can concentrate the proteins in the sampleby using a solid surface coated with a monoclonal antibody to capturethe protein. The recovered captured proteins can then be analyzed usingany suitable method described herein. The solid surface can be, e.g.,beads, such as magnetic beads, polystyrene beads, or gold beads, or inan array or a microarray format using a glass, a plastic or a siliconchip. Such protein capture can be also a part of a channel in amicrofluidic device.

Binding agents of use in this invention include an antibody, an antibodyfragment, or an antibody derivative (e.g., an aptamer) whichspecifically binds to a cognate filarial nematode protein. Specificbinding between two entities generally refers to an affinity of at least10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ aredesired to achieve specific binding.

When the binding agent is an antibody, the antibody can be produced bynatural (i.e., immunization) or partial or wholly synthetic means.Antibodies can be monoclonal or polyclonal and include commerciallyavailable antibodies. An antibody can be a member of any immunoglobulinclass, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.Bispecific and chimeric antibodies are also encompassed within the scopeof the present invention. Derivatives of the IgG class, however, aredesirable. Further, an antibody can be of human, mouse, rat, goat,sheep, rabbit, chicken, camel, or donkey origin or other species whichmay be used to produce native or human antibodies (i.e., recombinantbacteria, baculovirus or plants).

For example, naturally-produced monoclonal antibodies can be generatedusing classical cloning and cell fusion techniques or techniques whereinB-cells are captured and nucleic acids encoding a specific antibody areamplified (see, e.g., US 20060051348). In such methods, a collection ofproteins or an individual protein (e.g., a peptide or polypeptide) canbe used for the initial immunization and in the context of antibodyproduction is referred to herein as the antigen. The antigen of interestis typically administered (e.g., intraperitoneal injection) to wild-typeor inbred mice (e.g., BALB/c) or rats, rabbits, chickens, sheep, goats,or other animal species which can produce native or human antibodies.The antigen can be administered alone, or mixed with an adjuvant. Afterthe animal is boosted, for example, two or more times, the spleen orlarge lymph node, such as the popliteal in rat, is removed andsplenocytes or lymphocytes are isolated and fused with myeloma cellsusing well-known processes, for example, see Kohler & Milstein ((1975)Nature 256:495-497) or Harlow & Lane (Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory, New York (1988)). The resulting hybridcells are then cloned in the conventional manner, e.g., using limitingdilution, and the resulting clones, which produce the desired monoclonalantibodies, are cultured (see Stewart (2001) Monoclonal AntibodyProduction. In: Basic Methods in Antibody Production andCharacterization, Howard and Bethell (eds.), CRC Press, Boca Raton,Fla., pp. 51-67).

Alternatively, antibodies can be derived by a phage display method.Methods of producing phage display antibodies are known in the art,e.g., see Huse, et al. ((1989) Science 246(4935):1275-81). Selection ofantibodies is based on binding affinity to a protein or proteins ofinterest.

An antibody fragment encompasses at least a significant portion of thefull-length antibody's specific binding ability. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv,dsFv, diabody, Fd fragments or microbodies. An antibody fragment cancontain multiple chains which are linked together, for instance, bydisulfide linkages. A fragment can also optionally be a multi-molecularcomplex. A functional antibody fragment will typically include at leastabout 50 amino acid residues and more typically will include at leastabout 200 amino acid residues. The antibody fragment can be produced byany means. For instance, the antibody fragment can be enzymatically orchemically produced by fragmentation of an intact antibody or it can berecombinantly-produced from a gene encoding the partial antibodysequence. Alternatively, the antibody fragment can be wholly orpartially synthetically-produced.

Peptide aptamers which specifically bind to a protein are, in general,rationally designed or screened for in a library of aptamers (e.g.,provided by Aptanomics SA, Lyon, France). In general, peptide aptamersare synthetic recognition molecules whose design is based on thestructure of antibodies. Peptide aptamers are composed of a variablepeptide loop attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to that of an antibody (nanomolarrange).

Recombinant production of binding agents of this invention can beachieved using conventional molecular biology techniques andcommercially available expression systems. Furthermore, binding agentscan be produced using solid-phase techniques (see, e.g., Merrifield(1963) J. Am. Chem. Soc. 85:2149-2154; Seeberger (2003) Chem. Commun.(Camb) (10):1115-21). Protein synthesis can be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Boston, Mass.). Various fragments of a binding agent can bechemically-synthesized separately and combined using chemical methods toproduce a full-length molecule.

Moreover, combinatorial chemistry approaches can be used to producebinding agents (see, e.g., Lenssen, et al. (2002) Chembiochem.3(9):852-8; Khersonsky, et al. (2003) Curr. Top. Med. Chem. 3(6):617-43;Anthony-Cahill & Magliery (2002) Curr. Pharm. Biotechnol. 3(4):299-315).

The binding agents described herein can be labeled. In some embodiments,the binding agent is an antibody labeled by covalently linking theantibody to a direct or indirect label. A direct label can be defined asan entity, which in its natural state, is visible either to the nakedeye or with the aid of an optical filter and/or applied stimulation,e.g., ultraviolet light, to promote fluorescence. Examples of coloredlabels which can be used include metallic sol particles, gold solparticles, dye sol particles, dyed latex particles or dyes encapsulatedin liposomes. Other direct labels include radionuclides and fluorescentor luminescent moieties.

Indirect labels such as enzymes can also be used according to theinvention. Various enzymes are known for use as labels such as, forexample, alkaline phosphatase, horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. Fora detailed discussion of enzymes in immunoassays see Engvall (1980)Methods of Enzymology 70:419-439.

The proteins described herein (i.e., antibodies or antigens/epitopes)can be attached to a surface. Examples of useful surfaces on which theprotein can be attached for diagnostic purposes include nitrocellulose,PVDF, polystyrene, nylon or other suitable plastic. The surface orsupport may also be a porous support (see, e.g., U.S. Pat. No.7,939,342).

Further, the proteins of the invention can be attached to a particle orbead. For example, antibodies to the filarial nematode proteins or thefilarial nematode proteins themselves can be conjugated tosuperparamagnetic microparticles, e.g., as used in LUMINEX-basedmultiplex assays.

The filarial nematode proteins of this invention may be isolated and/orpurified or produced synthetically or using recombinant nucleic acidtechnology. The purification may be partial or substantial. Withreference to filarial nematode protein fragments, the term “fragment”refers to a protein having an amino acid sequence shorter than that ofthe proteins described herein. Preferably, such fragments are at least 5consecutive amino acids long or up to 35 amino acids long. In certainembodiments, the protein fragment includes at least one epitope. An“epitope” is a feature of a molecule, such as primary, secondary and/ortertiary peptide structure, and/or charge, that forms a site recognizedby an immunoglobulin, T cell receptor or HLA molecule. Alternatively, anepitope can be defined as a set of amino acid residues which is involvedin recognition by a particular immunoglobulin, or in the context of Tcells, those residues necessary for recognition by T cell receptorproteins and/or Major Histocompatibility Complex (MHC) receptors.

In some embodiments, the protein fragment of the invention is a fragmentof ALT2 comprising or consisting of the epitope of SEQ ID NO:121, inparticular epitopes of SEQ ID NO:78 or SEQ ID NO:98. In otherembodiments, the protein fragment of the invention is a fragment of ALT2comprising or consisting of the epitope of SEQ ID NO:122, in particularSEQ ID NO:79 or SEQ ID NO:99. In further embodiments, the proteinfragment of the invention is a fragment of HSP comprising or consistingof the epitope of SEQ ID NO:81 or SEQ ID NO:123, in particular SEQ IDNO:80 or SEQ ID NO:100. In certain embodiments, the protein fragment ofthe invention is a fragment of TSP comprising or consisting of theepitope of SEQ ID NO:82. In other embodiments, the protein fragment ofthe invention is a fragment of TPX2 comprising or consisting of theepitope of SEQ ID NO:83 or SEQ ID NO:124, in particular SEQ ID NO:84 orSEQ ID NO:101.

The fragments of the invention can be isolated, purified or otherwiseprepared/derived by human or non-human means. For example, epitopes canbe prepared by isolating the filarial nematode protein fragment from abacterial culture, or they can be synthesized in accordance withstandard protocols in the art. Synthetic epitopes can also be preparedfrom amino acid mimetics, such as D isomers of natural occurring L aminoacids or non-natural amino acids such as cyclohexylalanine.

In some embodiments, the filarial nematode protein or protein fragmentis conjugated or fused to a high molecular weight protein carrier tofacilitate antibody production. In some embodiments, the high molecularweight protein is bovine serum albumin, thyroglobulin, ovalbumin,fibrinogen, or keyhole limpet hemocyanin. A particularly preferredcarrier is keyhole limpet hemocyanin.

Any suitable immunoassay method may be used, including those which arecommercially available, to determine the level of at least one of thespecific filarial nematode proteins, protein fragments orprotective/neutralizing antibodies according to the invention. Extensivediscussion of the known immunoassay techniques is not required heresince these are known to those of skill in the art. Typical suitableimmunoassay techniques include sandwich enzyme-linked immunoassays(ELISA), radioimmunoassays (RIA), competitive binding assays,homogeneous assays, heterogeneous assays, etc. Various of the knownimmunoassay methods are reviewed, e.g., in Methods in Enzymology (1980)70:30-70 and 166-198.

In some embodiments, the immunoassay method or assay includes a doubleantibody technique for measuring the level of the filarial nematodeproteins or protein fragments in the biological sample. According tothis method one of the antibodies is a “capture” antibody and the otheris a “detector” antibody. The capture antibody is immobilized on a solidsupport which may be any of various types which are known in the artsuch as, for example, microtiter plate wells, beads, tubes and porousmaterials such as nylon, glass fibers and other polymeric materials. Inthis method, a solid support, e.g., microtiter plate wells, coated witha capture antibody, preferably monoclonal, raised against the particularprotein of interest, constitutes the solid phase. The biological sample,which may be diluted or not, typically at least 1, 2, 3, 4, 5, 10, ormore standards and controls are added to separate solid supports andincubated. When the protein of interest is present in the sample it iscaptured by the immobilized antibody which is specific for the proteinin question. After incubation and washing, a detector antibody, e.g., apolyclonal rabbit anti-marker protein antibody, is added to the solidsupport. The detector antibody binds to the protein bound to the captureantibody to form a sandwich structure. After incubation and washing ananti-IgG antibody, e.g., a polyclonal goat anti-rabbit IgG antibody,labeled with an enzyme such as horseradish peroxidase (HRP) is added tothe solid support. After incubation and washing a substrate for theenzyme is added to the solid support followed by incubation and theaddition of an acid solution to stop the enzymatic reaction.

The degree of enzymatic activity of immobilized enzyme is determined bymeasuring the optical density of the oxidized enzymatic product on thesolid support at the appropriate wavelength, e.g., 450 nm for HRP. Theabsorbance at the wavelength is proportional to the amount of protein ofinterest in the sample. A set of marker protein standards is used toprepare a standard curve of absorbance vs. filarial nematode proteinconcentration. This method is useful because test results can beprovided in 45 to 50 minutes and the method is both sensitive over theconcentration range of interest for each filarial nematode protein andis highly specific.

The standards may be positive samples containing various concentrationsof the protein to be detected to ensure that the reagents and conditionswork properly for each assay. The standards also typically include anegative control, e.g., for detection of contaminants. In some aspectsof the embodiments of the invention, the positive controls may betitrated to different concentrations, including non-detectable amountsand clearly detectable amounts, and in some aspects, also including asample that shows a signal at the threshold level of detection in thebiological sample.

The method of the invention can be carried out in various assay deviceformats including those described in U.S. Pat. Nos. 6,426,050,5,910,287, 6,229,603, and U.S. Pat. No. 6,232,114 to Aurora BiosciencesCorporation. The assay devices used according to the invention can bearranged to provide a quantitative or a qualitative (present/notpresent) result. In some embodiments, the method includes the use of amicrotiter plate or a microfluidic device format. The assays may also becarried out in automated immunoassay analyzers which are known in theart and which can carry out assays on a number of different samples.These automated analyzers include continuous/random access types.Examples of such systems are described in U.S. Pat. Nos. 5,207,987,5,518,688, 6,448,089, and 6,814,933. Various automated analyzers thatare commercially available include the OPUS® and OPUS MAGNUM® analyzers.

Another assay format which can be used according to the invention is arapid manual test which can be administered at the point-of-care at anylocation. Typically, such point-of-care assay devices will provide aresult which is either “positive,” i.e., showing the protein is present,or “negative” showing that the protein is absent. Typically, a controlshowing that the reagents worked in general is included with suchpoint-of-care system. Point-of-care systems, assays and devices havebeen well described for other purposes, such as pregnancy detection(see, e.g., U.S. Pat. Nos. 7,569,397 and 7,959,875). Accordingly, theinvention also provides devices, such as point-of-care test strips andmicrofluidic devices to perform the in vitro assays of the presentinvention.

It should be recognized also that the assay devices used according tothe invention can be provided to carry out one single assay for aparticular protein or to carry out a plurality of assays, from a singlevolume of body fluid, for a corresponding number of different filarialnematode proteins or antibodies thereto. In some embodiments, an assaydevice of the latter type is one which can provide a semi-quantitativeresult for the filarial nematode protein or antibodies measuredaccording to the invention, i.e., one or more of ALT2, TSP, VAL-1, TPX2,GST and HSP, or antibodies thereto. These devices typically are adaptedto provide a distinct visually detectable colored band at the locationwhere the particular protein of interest is located when theconcentration of the protein is above the threshold level. Foradditional detailed discussion of assay types which can be utilizedaccording to the invention as well as various assay formats andautomated analyzer apparatus see, e.g., U.S. Pat. No. 5,747,274.Filarial nematode protein detection can further be performed usingmultiplex technologies.

In other embodiments, the assays or immunoassays of the inventioninclude beads coated with a binding agent against a filarial nematodeprotein or a fragment thereof, or antibody. Commonly used arepolystyrene beads that can be labeled to establish a unique identity.Detection is performed by flow cytometry. Other types of bead-basedimmunoassays are known in the art, e.g., laser bead immunoassays andrelated magnetic bead assays (see, e.g., Fritzler, et al. (2009) ExpertOpinion on Medical Diagnostics 3:81-89).

The methods of the invention can be automated using robotics andcomputer directed systems. The biological sample can be injected into asystem, such as a microfluidic devise entirely run by a robotic stationfrom sample input to output of the result. The step of displaying theresult can also be automated and connected to the same system or in aremote system. Thus, the sample analysis can be performed in onelocation and the result analysis in another location, the onlyconnection being, e.g., an internet connection, wherein the analysis issubsequently displayed in a format suitable for either reading by ahealth professional or by a patient.

In certain embodiments, the presence of any one or any combination ofprotective/neutralizing antibodies described herein identifies a subjectas having been immunized with a multivalent immunogenic compositionagainst a filarial nematode. Thus, depending on antibody titer, thesubject may or may not receive additional booster vaccinations.

In some embodiments, the presence of any one or any combination of thefilarial nematode proteins described herein identifies a subject ashaving a filarial nematode infection. Thus, the subject is diagnosed ashaving filariasis and, in certain embodiments of this invention, treatedwith diethylcarbamazine, mebendazole, flubendazole, albendazole,ivermectin or a combination thereof. In one embodiment, the diagnosiscan be made if the presence of any one of the filarial nematode proteinsis detected in the subject's sample. In another embodiment, treatment isprescribed or administered if at least two of the filarial nematodeproteins are identified positively in the biological sample.

Kits provided according to this invention include one or more bindingagents, e.g., antibodies or antibody fragments, or filarial nematodeproteins, and optionally a device with a solid surface. In someembodiments, the solid surface is a bead, slide, assay plate (e.g., amultiwell plate) or a lateral flow device, to which the bindingagents/proteins are bound. In some embodiments, the kit further includesone or more standards or controls.

In some embodiments, the invention provides a microplate-based array formultiplex immunoassays. In accordance with some embodiments, each wellcan contain a single antibody against at least one of the listedfilarial nematode proteins. In other embodiments, each well contains anarray of antibodies against at least two or more of the listed filarialnematode proteins. In certain embodiments, each well of the plateincludes an antibody to two, three, four, or five of the followingproteins: ALT2, TSP, VAL-1, TPX2, GST and HSP. In particularembodiments, each well of the plate includes an antibody to each ofALT2, TSP, VAL-1, TPX2, GST and HSP.

In other embodiments, each well contains an array of at least two ormore of the filarial nematode proteins of this invention. In certainembodiments, each well of the plate includes two, three, four, or fiveof the following proteins: ALT2, TSP, VAL-1, TPX2, GST and HSP. Inparticular embodiments, each well of the plate includes each of ALT2,TSP, VAL-1, TPX2, GST and HSP.

In other embodiments, the invention provides simple to use point-of-carediagnostic test strips akin to pregnancy detection strips, wherein thestrip includes at least one antibody against at least one of the listedfilarial nematode proteins. In alternative embodiments, the inventionprovides simple to use point-of-care diagnostic test strips, wherein thestrip includes at least one of the instant filarial nematode proteins.

The test strip may include a positive and negative control to show theuser that the reagents work properly and/or that the sample has beenadded to the strip properly. The strips may be provided with or withouta casing and with or without additional reagents. Diagnostic test stripsfor lateral flow assays, such as the test strip assay described herein,may be constructed as described in the art, see, e.g., US 2010/0196200;US 2010/0129935; US 2009/0253119; and US 2009/0111171. Suitablematerials for test strips include, but are not limited to, materialsderived from cellulose, such as filter paper, chromatographic paper,nitrocellulose, and cellulose acetate, as well as materials made ofglass fibers, nylon, dacron, PVC, polyacrylamide, cross-linked dextran,agarose, polyacrylate, ceramic materials, and the like. The material ormaterials of the test strip may optionally be treated to modify theircapillary flow characteristics or the characteristics of the appliedsample. For example, the sample application region of the test strip maybe treated with buffers to correct the pH or specific gravity of anapplied sample, to ensure optimal test conditions.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1: Small Heat Shock Protein Vaccine

Parasites.

B. malayi L3s were obtained from the NIAID/NIH Filariasis ResearchReagent Resource Center (FR3) at the University of Georgia, Athens, Ga.

Human Sera Samples.

About 10 ml of blood samples were collected from the following clinicalgroups of subjects (1) Endemic normal (EN) subjects, these wereindividuals who were asymptomatic and non-microfilaraemic; (2)asymptomatic microfilaraemic subjects (Mf) who had circulatingmicrofilaria in their blood and were identified by microscopicexamination of their night blood smears; (3) Chronic Pathology (CP)patients include those subjects who exhibited lymph edema and otherchronic clinical symptoms of filariasis and (4) Non-endemic normalsubjects (NEN) who lived in non-endemic areas and had no circulatingparasites or antibodies and showed no evidence of any filarial disease.Sera were separated from these blood samples and were stored at −80° C.until use.

Expression and Purification of Recombinant B. malayi Heat Shock Protein.

To produce recombinant B. malayi small heat shock protein 12.6 (rBmHSP),the full-length gene sequence was cloned into pRSET-A (with anN-terminal hexahistidine tag) and was transformed into BL21(DE3)containing pLysS (Invitrogen, Carlsbad, Calif.) to minimize toxicity dueto the protein. When absorbance of the cultures reached 0.6 OD value, 1mM of IPTG (isopropyl thio-d-galactopyranoside) was added to thecultures and incubated for an additional 3 hours to induce geneexpression. After lysing the cells, total proteins were separated in a15% SDS-PAGE to confirm the expression of his-tagged protein.Subsequently, the histidine-tagged recombinant protein was purifiedusing an immobilized cobalt metal affinity column chromatography(Clontech, Mountain View, Calif.) as per the manufacturer'srecommendations. Recombinant protein was then separated in a 15%SDS-PAGE and stained with COOMASSIE brilliant blue R250. A single bandwas obtained after column purification.

Three-Dimensional Model of BmHSP.

A three-dimensional model of BmHSP protein was constructed by homologymodeling. BLAST sequence homology searches were performed to identifytemplate proteins in the PDB database. Human alpha-crystallin A, arecently crystallized protein, showed significant sequence identity andwas therefore chosen as the template for modeling BmHSP. Model buildingwas performed using MODELLER 9v6 (Sali & Blundell (1993) J. Mol. Biol.234:779-815). The 3-D structure obtained was subsequently validatedusing PROCHECK program (Laskowski, et al. (1993) J. Appl. Cryst.26:283-29). The best model predicted by PROCHECK had a score of −0.46and was chosen for further modeling and for generating the 3-D structureusing Rasmol program.

Analysis of the Structure of BmHSP.

The secondary structure and protein-protein interaction site of BmHSPwas predicted at PDBsum and the Predict Protein E-mail server at theEuropean Molecular Biology Laboratory, Heidelberg (Roos, et al. (1995)Parasitol. Today 11:148-150). Motif scanning was carried out via PROSITEpattern analysis to identify the functional motifs in BmHSP. B-cell,T-cell and CTL epitopes in BmHSP sequences were predicted using ImmuneEpitope Database and Analysis Resource (lEDB).

Phylogenetic Analysis of BmHSP.

Amino acid sequences of BmHSP were compared with members of other smallheat shock family of proteins from different organisms. The followingsequences were analyzed. Accession numbers are given in parenthesis.Aconthocheilonema vitae (CAA48631); Archaeoglobus fulgidus (028308);Artibeus jamaicensis (P02482); Aspergillus fumigatus (Q4WV00);Arabidopsis thaliana (081822); Artemia persimilis (DQ310578);Azotobacter vinelandii (P96193); Brugia pahangi (CAA61152), Brugiamalayi (AAU04396); Buchnera aphidicola (P57640); Bombyx mori(AF315318_1); Bradyrhizobium japonicum (P70918); Caenorhabditis elegans(Q7JP52); Coccidioides immitis (Q1E6R4); Carica papaya (Q69BI7);Caenorhabditis remanei (AAZ42349); Dictyostelium discoideum (Q54191);Escherichia coli (ibpA; POC054); Escherichia coli (ibpB; POC058); Homosapiens (P02489); Haemonchus contortus (AAN05752); Lygodactyluspicturatus (Q6EWI0); Onchocercara volvulus (CAA48633), Ostertagiaostergi (CAG25499); Macaca mulatta (P02488); Mycobacterium tuberculosis(POA5B7); Mus musculus (AAA37861); Nippostrongylus brasiliensis(BAI81970); Plasmodium falciparum (Q8IB02); Rattus (CAA42910);Saccharomyces cerevisiae (P15992); Solanum lycopersicum (082545);Streptococcus thermophilus (P80485); Trichinella spiralis (ABJ55914);Trypanosoma brucei (Q57V53); Toxoplasma gondii (Q6DUA8). Thealpha-crystallin domain from all sHSP sequences were aligned usingClustalW algorithm and the data set were used to build a phylogenetictree with the PHYLIP software. The trees were made using the neighborjoining method, with Poisson-corrected amino acid distances.

Chaperone Assay.

One of the typical characteristics of chaperone is that they can bind toand protect cellular proteins from heat damage. When proteins areexposed to heat damage, they aggregate (thermal aggregation). Chaperonesprevent this aggregation. To determine whether BmHSP could preventthermal aggregation, a citruline synthase (CS) (Sigma, St. Louis, Mo.)thermal aggregation assay was used. CS was selected because this proteinis highly sensitive to heat denaturation. An established method was used(Gnanasekar, et al. (2009) Biochem. Biophys. Res. Commun. 386:333-337).Briefly, 1 μM of CS was exposed to 45° C. in the presence or absence ofBmHSP (2 IiM) suspended in 50 mM of sodium phosphate pH 7.4 buffercontaining 100 mM NaCl. BSA was used as a control. CS was incubated withBmHSP at a molar ratio of (1:2) for various time intervals from 0 to 40minutes. Thermal denaturation (aggregation) was monitoredspectrophotometrically at 360 nm.

In Vitro Peptide Binding Assay for Chaperone Activity.

Another characteristic of heat shock proteins is that they can bind to avariety of proteins. To determine whether BmHSP also possesses thisfunction, CS and another protein, luciferase, were chemically denaturedwith 6M guanidine hydrochloride according to known methods (Gnanasekar,et al. (2009) supra). Native and chemically denatured proteins were thencoated onto 96-well plates overnight at 4° C. After washing with PBS,wells were blocked with 3% BSA at room temperature. Following furtherwashing, wells were incubated with his-tagged rBmHSP for 1 hour at 37°C. After washing again with PBS, optimally diluted anti-his-tagged HRPconjugate was added and incubated at 37° C. for 1 hour. After finalwashing, color was developed with ABTS[2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] and OD wasmeasured at 405 nm.

Anti-BmHSP Antibody Levels in Human Sera.

A total of 20 sera samples belonging to different clinical groups suchas Mf, CP, EN and NEN were analyzed for the presence and titer ofanti-BmHSP IgG antibodies using an indirect ELISA (Cheirmaraj, et al.(1992) J. Trop. Med. Hyg. 95:47-51). Briefly, wells of a 96-wellmicrotiter plate were coated with rBmHSP (1 μg/ml) in carbonate buffer,pH 9.6, overnight at 4° C. and blocked with 3% BSA for 1 hour at 37° C.Sera samples were added to the wells and the plates were incubatedovernight at 4° C. After washing the wells, HRP-labeled mouse anti-humanIgG was added (1:5000) and incubated further for 1 hour at 37° C. Colorwas developed using ABTS substrate. Absorbance was measured at 405 nm ina microplate reader (BIO-RAD, Hercules, Calif.). The isotype ofanti-BmHSP IgG antibodies in the sera of subjects was also determinedusing an isotype-specific ELISA. Biotinylated mouse monoclonal antihumanIgG1, IgG2, IgG3 and IgG4 were used as the secondary antibodies andcolor was developed with avidin-HRP conjugate (Sigma, St. Louis, Mo.) asthe secondary antibodies.

Cloning of Codon-Optimized BmHSP into pVAX Vector for DNA Vaccine.

Codon-optimized Bmhsp genes were cloned into eukaryotic expressionvector pVAX (Invitrogen) using insert-specific primers (forward primer,5′-CGC GGA TCC ATG GAA GAG AAG GTG GTG-3′ (SEQ ID NO:1) containing BamHIsite and reverse primer, 5′-CCG GAA TTC TCA CTT GTC GTT GGT G-3′ (SEQ IDNO:2) containing EcoRI site). PCR parameters were as follows: 94° C. ofdenaturation for 30 seconds, 50° C. of primer annealing for 30 seconds,72° C. of primer extension for 30 seconds for 30 cycles; and a finalextension of 5 minutes was performed at 72° C. Insert DNA was sequencedto ensure authenticity of the cloned nucleotide sequence on bothstrands. Plasmids were maintained and propagated in E. coli TOP10F′cells. Subsequently, plasmids were purified using endotoxin-free plasmidextraction kit (Qiagen, Hilden, Germany). DNA was analyzed by agarosegel electrophoresis and quantified by spectrophotometry (OD 260/280,ratio>1.8).

Immunization of Mice.

Six-week-old male Balb/c mice purchased from Charles River Laboratorieswere used in these experiments. Humane use of animals in this study andthe protocol was approved by the IACUC committee at the College ofMedicine, University of Illinois Rockford. Each group was composed offive (5) mice and all mice were immunized intraperitoneally using threedifferent immunization regimens. Group A mice were immunized using aprime-boost regimen. Mice were primed twice at two-week intervals with100 μg of endotoxin-free, codon-optimized pVAX Bmhsp DNA suspended in 50μl volume. Following priming, all mice received two booster doses of 15μg of rBmHSP protein (50 μl each) suspended in alum at two weeksinterval. Group B mice were immunized with rBmHSP protein alone. Thesemice received four doses of 15 μg of rBmHSP protein suspended in alumgiven at two-week intervals. Group C mice were immunized with DNA alone.These mice received four doses of 100 μg of pVAX Bmhsp DNA given attwo-week intervals. Group D animals received 100 μg of pVAX vectorcontrol and adjuvant at the same interval and remained as negativecontrols. Blood samples were collected from each mouse beforeimmunization and one month after the last booster dose. After separatingthe sera, titer of circulating anti-BmHSP IgG antibodies and therespective isotypes were determined. Sera that showed high titer ofantibodies against BmHSP were used in the Antibody Dependent CellularCytotoxicity (ADCC) assay described herein.

Anti-BmHSP Antibody Levels in the Sera of Mice.

Anti-BmHSP IgG antibody levels in the sera of immunized and controlgroups of mice were determined using an indirect ELISA (Veerapathran, etal. (2009) PLoS Negl. Trop. Dis. 3:e457). IgG1, IgG2a, IgG2b and IgG3anti-BmHSP antibody levels were also determined using a mouse antibodyisotyping ELISA kit (ThermoFisher Scientific, Rockford, Ill.). Color wasdeveloped with ABTS (2,2′-azinobis (3-ethyl benzothiazoline-6-sulfonicacid) chromogen substrate and the absorbance was measured at 405 nm inan ELISA reader (BIO-RAD).

Depletion of Anti-BmHSP Antibodies from Human and Mice Sera.

Anti-BmHSP antibodies were depleted from pooled sera of EN subjects andimmunized mice by incubating the pooled sera with cobalt IMAC resincoupled with his-tagged rBmHSP according to established methods(Veerapathran, et al. (2009) supra). Briefly, 1 mg of his-tagged rBmHSPwas coupled to 2 ml bed volume of IMAC resin for 2 hours at 37° C. Afterwashing the resin once with 10 ml of PBS (pH. 8), 200 μl of pooled serawas added and incubated overnight at 4° C. After incubation, the resinmixture was centrifuged for 2 minutes at 750 rpm and the supernatant wascollected. Depletion of anti-BmHSP antibodies in the supernatant wasconfirmed by ELISA as described herein.

Anti-BmHSP IgG1, anti-BmHSP IgG2a, anti-BmHSP IgG2b, anti-BmHSP IgG3 andanti-BmHSP IgG4 antibodies from pooled sera of EN subjects and pooledsera of immunized mice were depleted using NHS (N-hydroxysuccinimidyl)resin (Thermo fisher scientific). Briefly, 1 μg of respective monoclonalantibodies were coupled to NHS resin column. After washing the resintwice with PBS (pH. 8), 100 μl of sera were passed through the column.The flow through was collected as the antibody depleted sera. Depletionof the specific isotype of antibody was confirmed by an isotype-specificELISA as described herein. After washing the column three times with PBS(pH 7.4), bound antibodies were eluted using Glycine-HCl buffer (pH 2.7)from the resin and the pH was adjusted to 7.4 with 1 M Tris buffer (pH8). The recovered elute contained the specific antibody as confirmedagain by an ELISA. The antibody depleted sera was also reconstitutedwith the eluted antibodies. An aliquot of depleted sera wasreconstituted with the eluted antibodies to its original concentrationusing values determined by an earlier ELISA on the neat serum samples.Antibody depleted sera, eluted antibodies and reconstituted sera sampleswere then used in an ADCC assay.

Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay.

In vitro ADCC assay was performed according to known methods(Chandrasekhar, et al. (1985) Parasite Immunol. 7:633-641). Briefly, ten(10) L3 of B. malayi were incubated with 2×10⁵ peritoneal cells (PEC)collected from normal mice, 50 μl of pooled mouse sera samples and 50 μlof RPMI 1640 media in a 96-well culture plate (Thermo FisherScientific). After 48 hours of incubation at 37° C. and 5% CO₂, larvalviability was determined at 400× using a light microscope. Larvae thatwere limpid and damaged were counted as dead. In addition, dead larvaealso had clumps of cells adhered to it and were more transparent thanlive. Larvae that were active, coiled and translucent were counted aslive. ADCC was estimated as the percent larval death calculated usingthe formula:

Number of Dead larvae÷Total Number of Larvae×100.

ADCC assay was also performed with pooled human sera samples asdescribed herein except that the human sera samples were incubated with2×10⁵ PBMCs collected from normal health subjects and 6-12 B. malayi L3for 48 hours at 37° C. and 5% CO₂. Larval viability and death weredetermined as described above.

Protection Studies in Mice.

Vaccine potential of BmHSP was evaluated in a mouse model of challengeinfection. Mice were immunized as described above using prime-boost, DNAalone or protein alone approach. Vector and alum group served asnegative controls. Immunized and control animals were challenged using amicropore chamber method as known in the art (Abraham, et al. (1986)Immunology 57:165-169). Briefly, micropore chambers were assembled using14×2 mm PLEXIGLASS (acrylic) rings (Millipore Corporations, Bedford,Mass.) and 5.0 μm NUCLEOPORE polycarbonate membranes (MilliporeCorporations). The membranes were attached to the PLEXIGLASS rings withcyanoacrylic adhesive and dental cement. The chambers were immersedovernight at 37° C. in sterile RPMI medium containing gentamycin andantimycotic solution. Before challenge experiments, 20 live, infectiveL3s suspended in RPMI 1640 medium supplemented with 15% heat-inactivatedfetal calf serum (FCS) were introduced into the micropore chambers andthe opening was sealed with dental cement. Micropore chamber containingthe L3s were then surgically implanted into the peritoneal cavity ofeach mice under anesthesia. Aseptic conditions were followed for thesurgical procedures. After 48 hours of implantation, animals weresacrificed and the chambers were recovered from peritoneal cavity.Contents of each chamber were emptied and larvae were examinedmicroscopically for adherence of cells and for larval death. Dead andlive larvae were identified as described above under ADCC. Thepercentage of protection was expressed as the number of deadparasites÷number of total parasites recovered×100.

Splenocyte Proliferation Assay.

Spleens were collected from all mice from the above experiment andsingle-cell suspension of spleen cells was prepared. Approximately 2×10⁵cells/well suspended in complete RPMI 1640 medium supplemented with 10%heat-inactivated FCS were incubated at 37° C. and 5% CO₂ for 72 hourswith rBmHSP (1 μg/ml), ConA (1 Hg/ml) or with medium alone. Afterincubation, cell proliferation was determined using cell counting kit(CCK-8) purchased from Dojindo Molecular Technologies, Inc.(Gaithersburg, Md.). Stimulation index of spleen cell proliferation wascalculated using the formula: Absorbance of stimulated cells÷Absorbanceof unstimulated cells.

Cytokine Analysis.

Spleen cells from immunized and control mice were cultured at 37° C. and5% CO₂ for 72 hours with rBmHSP (1 μg/ml), ConA (1 μg/ml) or with mediumalone as described above. After 72 hours, culture supernatants and cellpellets were collected separately for cytokines analysis. For measuringcytokine mRNA, cell pellets were suspended in TRIZOL (phenol,guanidinium and thiocyanate) reagent (GIBCO-BRL, Life technologies,Carlsbad, Calif.) and total RNA was extracted as per the manufacturer'sinstructions. After ethanol washes, RNA pellets were dissolved inRNAse-free water (Sigma) and treated with DNase I before determiningtotal RNA concentration using a Beckman spectrophotometer at 260 nm.Reverse transcription of total RNA was performed using first strand cDNAsynthesis kit (SABiosciences, Frederick, Md.) as per manufacturer'srecommendations. Relative quantification of the expression of genes ofinterest was measured in an Applied BioSystems 7300 real-time PCRmachine (Applied BioSystems, Foster City, Calif.). PCR amplificationswere performed with the LIGHTCYCLER-DNA SYBR Green (cyanine dye) mix(SAbiosciences). The reaction was performed using the following PCRconditions: 15 minutes activation step at 95° C. for one cycle, 15seconds denaturation step at 95° C., annealing of primers for 20 secondsat 50° C. and elongation step for 15 seconds at 72° C. DNA was amplifiedfor 50 cycles. The fluorescent DNA binding dye SYBR Green (cyanine dye)was monitored. RT-PCR data array set was generated and analyzed usingSABiosciences web-based data analysis system.

Culture supernatants were then collected from splenocyte cultures 72hours after incubation with rBmHSP (1 μg/ml), ConA (1 μg/ml) or withmedium alone. Secreted levels of IL-2, IL-4, IFN-γ and IL-10 protein inthe culture supernatants were determined using a sandwich ELISA kitpurchased from ThermoFisher Scientific. Concentration of each cytokinewas determined from a standard curve plotted using recombinant mouseIL-2, IL-4, IFN-γ or IL-10.

Statistical Analysis. Statistical analysis was performed using XL STATsoftware v.7.5.2 (Kovach Computing Services, Anglesey, UK). Statisticalsignificance between comparable groups was estimated using appropriatenon-parametric tests, with the level of significance set at p<0.05.

Expression of Recombinant BmHSP12.6 (BmHSP).

BmHSP was cloned in pRSET A vector and was expressed as ahistidine-tagged (his-tagged) fusion protein in E. coli BL21 (DE3)PLysS.Recombinant BmHSP protein was subsequently purified using IMAC column.The molecular mass of the purified recombinant his-tag fusion proteinwas found to be approximately 18 kDa. The column-purified recombinantprotein appeared as a single band in SDS-PAGE.

Predicted Three-Dimensional Structure of BmHSP.

Amino acid sequences of the human alpha crystalline A chain share 42%similarity with BmHSP. Since crystal structure of the human alphacrystalline A chain is already available, this was used that as atemplate to model the putative structure of BmHSP using the Modeller 9v6program. PROCHECK analysis was used to select the best model that showeda score of −0.41 compared to the template (Laskowski, et al. (1993) J.Appl. Cryst. 26:283-29). A Ramachandran plot analysis was also performedon the BmHSP sequence. These analyses showed that 92% of residues werein the most favorable region with no steric hindrance. About 6.7%residues were found in the additional allowed region. Models that showedover 90% residues in the most favored regions were predicted as the mostideal three-dimensional model as predicted by the Ramachandran plot(Balazs, et al. (2001) Protein Eng. Des. Sci. 14:875-880). Secondarystructure prediction analysis was also performed on BmHSP protein usingPDBsum server at EMBL. This analysis showed that each alpha-crystallinedomain of BmHSP monomer had an immunoglobulin core composed of sevenβ-strands arranged in two anti-parallel sheets. The secondary structureprediction of BmHSP showed two sheets, four beta hairpins, one betabulge, seven strands, two helices, seven beta turns and one gamma turnin the structure of BmHSP.

Previous studies showed that BmHSP binds to human IL-10 receptor I αchain (Gnanasekar, et al. (2008) Mol. Biochem. Parasit. 159:98-103). Toidentify the IL-10 receptor binding site on BmHSP, a predictiveprotein-protein interaction analysis was performed (Ofran & Rost (2007)Bioinformatics 23:e13-e16). Results from the prediction analysis showedthat the N-terminal fragment of BmHSP (amino acids from Met1 to Asn26)had a strong protein-protein interaction region. Further sequenceanalysis of this region showed that the amino acid sequences from Val5to Glu42 had significant sequence identity to human IL-10R bindingregion of human IL-10. These findings confirm that the N-terminal regionof BmHSP may be involved in the binding of BmHSP to human IL-10 receptorI α chain.

Motif and Phylogenetic Analysis on BmHSP.

Motif analysis performed at PROSITE showed several putativepost-translation modification sites such as N-glycosylation sites(residues 11 to 14 and 98 to 101), protein kinase-c phosphorylationsites (residues 83 to 85 and 100 to 102), casein kinase IIphosphorylation sites (residues 68 to 71 and 88 to 91) andN-myristylation sites (residues 40 to 45) on BmHSP. Similar motifs werealso observed in human IL-10 further indicating that BmHSP may mimichuman IL-10 function (Gnansekar, et al. (2008) supra). Epitope mappingon BmHSP revealed the presence of B-cell, T-cell and CTL epitoperegions, indicating that BmHSP is potentially a highly immunogenicprotein (Table 2).

TABLE 2 Position of  Epitope in SEQ Epitope  the Amino  Peptide IDPredicted Acid Sequence Sequence NO: B-Cell 12-23 WSAEQWDWPLQH  3Epitopes 26-35 EVIKTNTNDK  4 67-74 SRAEHYGE  5 84-94 KLPSDVDTKTL  6T-Cell 45-53 FTPKEIEVK  7 Epitopes 50-57 IEVKVAGD  8 38-45 VGLDASFF  939-47 GLDASFFTP 10 52-60 VKVAGDNLV 11 84-92 KLPSDVDTK 12  92-100KTLTSNLTK 13 27-35 VIKTNTNDK 14 90-98 DTKTLTSNL 15 44-52 FFTPKEIEV 1638-46 VGLDASFFT 17 100-108 KRGHLVIAA 18 73-81 GEIKREISR 19 43-51SFFTPKEIE 20 CTL 78-86 REISRTYKL 21 Epitopes 74-82 GEIKREISR 22 70-78AEHYGEIKR 23

A phylogenetic analysis performed using representative sHSP sequencesfrom different groups of organisms showed that BmHSP, C. elegans HSP andC. remani HSP form a monophyletic group, separated from the other groupsof organisms.

BmHSP is a Chaperone.

Most of the heat shock proteins reported to date have chaperonefunction. To determine whether BmHSP also has similar chaperonefunction, a thermal aggregation reaction was performed using a modelsubstrate, Citrulline synthase (CS). Incubation of CS at 42° C. resultedin unfolding of the protein and subsequent aggregation within 10minutes. Addition of BmHSP to CS protein (at a molar ratio of 1:2),before the heat treatment, significantly (P<0.01) inhibited the thermalaggregation of CS protein. A non-chaperone control protein, BSA, had noeffect on the heat-induced aggregation of CS protein.

Another function of chaperone proteins is that they can specificallybind to denatured proteins. To determine whether BmHSP can specificallybind to denatured proteins, rBmHSP was incubated with native anddenatured CS or native and denatured luciferase substrates. Thesestudies showed that rBmHSP preferentially bound to denatured proteinsubstrates compared to native or control protein. These findings thusconfirmed that BmHSP can act as a molecular chaperone potentiallyprotecting the parasite cellular proteins from the damaging effects ofthe host.

Antibody Responses in Human.

The results presented herein indicate that BmHSP has several T-cell andB-cell epitopes. Therefore, it was evaluated whether filariasis-infectedindividuals carry antibodies to BmHSP. Accordingly, the titer ofanti-BmHSP IgG antibodies in the sera of EN, CP, Mf and NEN subjects wasmeasured. The results showed that the EN subjects had the highest levelsof anti-BmHSP antibodies (p<0.001). Subsequent isotype analysis of theIgG antibodies showed that compared to the infected groups (Mf and CP)of individuals, sera from EN subjects had high titers of IgG1 and IgG3anti-BmHSP antibodies. Mf carriers had only significant levels ofanti-BmHSP IgG2 antibodies in their sera. Similarly, CP individuals hadonly significant levels of anti-BmHSP IgG4 antibodies in their sera.Anti-BmHSP IgG1 and IgG3 levels were very low in the sera of these Mfand CP individuals. Anti-BmHSP antibodies were not detectable in thesera of NEN subjects.

Results of ADCC Assay.

Since antibodies to BmHSP were present in all infected groups ofindividuals (Mf and CP) and EN subjects, it was determined whether theseantibodies were functional. Using an antibody-dependent cellcytotoxicity assay, it was tested if anti-BmHSP12.6 IgG antibodies hadany protective function against B. malayi. These studies showed thatpooled EN sera promoted adherence of PBMC's to L3 and inducedsignificant (77.37%) death of B. malayi L3s in vitro (Table 3), whereas,pooled sera from Mf and CP failed to participate in the ADCC function.These findings indicated that EN sera have anti-parasitic activities. Todetermine if this function is associated with antibodies, antibodydepletion studies were performed. Depletion of anti-BmHSP antibodiesfrom EN sera resulted in significant reduction (21.42%) in larval death(Table 3) confirming that anti-BmHSP antibodies in the sera of ENsubjects, but not Mf or CP subjects, participate in larval killing.

TABLE 3 Dead Live Total % Larval Death Groups L3 L3 L3 (Mean ± SD*)Endemic Normal (EN) sera 5 1 6 77.37 ± 8.41 5 2 7 EN sera depleted ofanti- 2 5 7  21.42 ± 10.12 rBmHSP antibodies 1 6 7 Non-Endemic Normal(NEN) 1 5 6 {grave over ( )}19.44 ± 3.92 sera 2 7 9 *Values representmean ± SD of three wells.

Further depletion studies showed that the anti-parasitic effect ofanti-BmHSP antibodies was associated with IgG1 isotype of antibodies.Depletion of IgG1 antibodies from EN sera significantly (40%) inhibitedthe ADCC function (Table 4). Reconstitution of anti-BmHSP antibodydepleted EN sera with eluted anti-BmHSP IgG1 antibodies regained theADCC function (Table 3). These findings thus indicated that anti-BmHSPIgG1 antibodies are critical for ADCC function.

TABLE 4 EN Sera % Larval Death Neat Sera 72 Depleted of: IgG1 40 IgG271.43 IgG3 60 IgG4 62.5 Reconstituted IgG1 70 with: IgG2 69.23 IgG366.67 IgG4 54.55 Values represent mean of three wells.

Antibody Responses in Mice.

Mice immunized with rBmHSP developed significant levels of anti-BmHSPIgG antibodies. More specifically, prime-boost vaccine regimen inducedsignificantly higher titer of IgG antibodies compared to DNA vaccinealone group (p<0.05). However, rBmHSP protein vaccine induced thehighest IgG antibody titer. Analysis of the isotype of anti-BmHSP IgGantibodies showed that predominantly IgG1, IgG2a and IgG2b anti-BmHSPantibodies were present in the sera of vaccinated animals. The ADCCassay was also performed with mouse sera. These studies showed that serafrom BmHSP-vaccinated mice promoted adherence of peritoneal exudatecells to L3 and participated in ADCC function (83.02% larval killing)compared to control sera (13%) (p<0.002) (Table 5).

TABLE 5 Immunization Regimen % Larval Death Bmhsp DNA prime and rBmHSPprotein boost 83.02 ± 3.62 Bmhsp DNA  43.7 ± 8.12 rBmHSP protein 55.08 ±1.15 pVAX & alum control   13 ± 2.35 Values represent mean ± SD of threewells.

Similar to human sera, individual isotypes of IgG antibodies weredepleted from the sera of vaccinated mice to determine the isotype ofanti-BmHSP antibodies that participate in the ADCC function. Resultsfrom these studies showed that, similar to that observed with EN sera,anti-BmHSP IgG1 antibodies were involved in ADCC-mediated killing of L3in mice as well (Table 6).

TABLE 6 Immunized Mice Sera % Larval Death Neat Sera of BmHSPprime-boost 80.16 Depleted of: IgG1 37 IgG2a 72 IgG2b 71 IgG3 80Reconstituted with: IgG1 80 IgG2a 63 IgG2b 87 IgG3 71 Values representmean of three wells.

Vaccine Potential of BmHSP in Mice.

Vaccine potential of BmHSP was assessed in Balb/c mice using a microporechamber method. Results showed that mice immunized using the prime-boostvaccination regimen and protein vaccine of BmHSP exhibited nearly 72%and 58% mortality, respectively, of L3s implanted into the peritonealcavity of the immunized mice (Table 7). While chambers implanted in thecontrol groups of animals showed only 7% mortality of the parasite, thedifference between the protection of control group of mice andvaccinated mice was significant (P<0.001). On the other hand, miceimmunized by DNA vaccine alone induced only 31% protection. Thus, theprime-boost vaccination regimen appeared to be highly efficient inconferring vaccine-induced protection against a challenge infectioncompared to DNA alone or protein alone immunization protocols.

TABLE 7 Immunization Regimen % Larval Death Bmhsp DNA prime and rBmHSPprotein boost  72 ± 10.22 Bmhsp DNA 31 ± 5.23 rBmHSP12.6 protein 58 ±7.76 pVAX & alum control 7 ± 5.2 Values represent mean ± SD. N = 5. Datais from one of two similar experiments showing comparable results.

Immune Responses in BmHSP Vaccinated Mice.

To determine cellular immune responses to BmHSP in the vaccinated mice,spleen cells collected from vaccinated and control mice were cultured inthe presence of rBmHSP protein and their proliferative responses andcytokine profiles were evaluated. Proliferative response of spleen cellsfrom animals immunized with the prime-boost vaccine regimen wassignificantly (P>0.05) higher (stimulation index of 3.35±0.176) comparedto rBmHSP protein alone vaccination group (stimulation index of2.22±0.018) or Bmhsp DNA vaccination alone group (stimulation index of3.53±0.102). Spleen cells from the control group of animals failed toproliferate in response to rBmHSP (stimulation index of 0.98±0.013) andwas similar to media alone controls. Since the spleen cells fromvaccinated animals were proliferating significantly to recall responseto rBmHSP, levels of cytokines in the culture supernatants weremeasured. These results showed that IFN-γ was the predominant cytokinesecreted by spleen cells from vaccinated animals at 72 hours afterstimulation with rBmHSP. A real time-PCR cytokine gene array wasperformed on mRNA collected from the spleen cells stimulated withrBmHSP. These results showed that both Th1 (IFN-γ, CD-28, IL-12, IL-2)and Th2 (IL-4, IL-5, IL-1R) cytokine genes were significantly increasedin vaccinated animals.

Example 2: rBmALT2+rBmHSP Multivalent Immunogenic Composition

Parasite.

Brugia malayi L3s were obtained from the NIAID/NIH Filariasis ResearchReagent Resource Center (FR3) at the University of Georgia, Athens, Ga.

Construction of Monovalent and Multivalent DNA Vaccines.

Monovalent DNA vaccine was composed of Bmhsp or Bmalt2 in pVAX1 vector.To prepare the monovalent vaccine, codon optimized Bmhsp or BmALT2 geneswere cloned into the eukaryotic expression vector pVAX1 (Invitrogen,Carlsbad, Calif.) using insert-specific primers (Gnanasekar, et al.(2004) supra). The multivalent immunogenic composition was composed ofBmhsp and Bmalt2 genes in the same pVAX1 vector. Codon optimized Bmhspgene was first cloned into pVAX1 vector with no stop codon in thereverse primer (5′-CCG GAA TTC TCA CTT GTC GTT GGT G-3′; SEQ ID NO:24)but contained a PstI site. Codon optimized Bmalt2 gene was then insertedinto this clone using gene specific primers (Gnanasekar, et al. (2004)supra). PCR parameters for all the three constructs were: 94° C.denaturation for 30 seconds, 50° C. primer annealing for 30 seconds, 72°C. primer extension for 30 seconds for 30 cycles; a final extension of 5minutes was performed at 72° C. Insert DNA was finally sequenced toensure authenticity of the cloned nucleotide sequence on both strands.Plasmids were maintained and propagated in E. coli TOP10F′ cells.Plasmids were purified using endotoxin-free plasmid extraction kit(Qiagen, Valencia, Calif.). DNA was analyzed by agarose gelelectrophoresis and quantified in a spectrophotometer (OD 260/280, ratio>1.8).

Expression and Purification of Recombinant Proteins.

All the genes were cloned in pRSET-A vector (with an N-terminalhexahistidine tag) to produce recombinant proteins. Bmhsp and Bmalt2constructs were transformed into BL21(DE3) containing pLysS E. coli host(Invitrogen) to minimize toxicity due to the protein. When absorbance ofthe cultures reached 0.6 OD value, 1 mM of IPTG (isopropylthio-d-galacto pyranoside) was added to the cultures and incubated foran additional 3 hours to induce the gene expression. After lysing thecells, total proteins were separated in 15% and 12% SDS-PAGE to confirmthe expression of his-tag recombinant BmHSP (rBmHSP) and rBmALT2proteins. The recombinant proteins were then purified using animmobilized cobalt metal affinity column chromatography (Clontech,Mountain View, Calif.) as per the manufacturer's recommendations.Recombinant proteins were then separated in SDS-PAGE and stained withCOOMASSIE brilliant blue R250 and silver stain. These studies showedthat a single band was obtained after column purification. Endotoxins ifany in the recombinant preparations were removed by passing therecombinant proteins through polymyxin B affinity columns (Thermo FisherScientific, Rockford, Ill.) and the levels of endotoxin in the finalpreparations were determined using an E-TOXATE kit (Sigma, St Louis,Mo.) as per manufacturer's instructions. Endotoxin levels were belowdetection limits in these recombinant protein preparations.

Immunization of Mice.

Six-weeks old male Balb/c mice purchased from Charles River Laboratorieswere used in these experiments. Humane use of animals in this study andthe protocol was approved by the IACUC committee at the College ofMedicine, University of Illinois Rockford. Mice were divided into four(4) groups of five (5) animals each. All mice were immunizedsubcutaneously using a DNA prime-protein boost vaccine regimen. Allexperimental groups of mice were primed with two injections ofendotoxin-free codon optimized DNA given in 50 μl volume and boostedwith two doses of recombinant proteins suspended in alum (50 μl each)given at two weeks interval.

Group A mice were primed with 100 μg of pVAXBmhsp and boosted with 15 μgof rBmHSP; Group B mice were primed with 100 μg of pVAX Bmalt2 andboosted with 15 μg of rBmALT2; Group C mice were primed with 100 μg ofpVAXBmhsp/Bmalt2 DNA and boosted with 15 μg of rBmHSP and 15 μg ofrBmALT2. Group D mice received 100 μg of pVAX1 vector plus 50 μl of alumand served as controls. Blood samples were collected from each mousebefore immunization and one month after the last booster dose. Sera wereseparated and stored at −80° C.

Evaluation of Antibody Responses in Mice.

Levels of anti-BmHSP and anti-BmALT2 antibodies were measured in thesera of immunized and control groups of mice using an indirect ELISAaccording to established methods (Veerapathran, et al. (2009) supra;Gnanasekar, et al. (2004) supra). Briefly, wells of 96-well microtiterplates were coated with rBmHSP, rBmALT2 or rBmHSP (1 μg/ml) in carbonatebuffer (pH 9.6) overnight at 4° C. After washing the wells, unboundsites were blocked with 3% BSA for 1 hour at 37° C. Diluted sera sampleswere then added to the wells and incubated further overnight at 4° C.After washing the wells, HRP-labelled rabbit anti-mouse IgG was added(1:5000) and incubated further for 1 hour at 37° C. Color was developedusing ABTS (2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid))substrate. Absorbance was measured at 405 nm in a microplate reader(BIO-RAD, Hercules, Calif.).

Protection Studies in Mice.

Vaccine potential of the monovalent and multivalent immunogeniccomposition formulations were then evaluated in a mice model. Mice wereimmunized as described above using the prime boost approach. Vector plusalum group served as negative controls. Immunized and control animalswere challenged using a micropore chamber method known in the art(Abraham, et al. (1989) Am. J. Trop. Med. Hyg. 40(6):598-604). Briefly,micropore chambers were assembled using 14×2 mm PLEXIGLASS (acrylic)rings (Millipore Corporations, Bedford, Mass.) and 5.0 μm NUCLEOPOREpolycarbonate membranes (Millipore Corporations) that were attached tothe PLEXIGLASS (acrylic) rings with cyanoacrylic adhesive and dentalcement. The chambers were immersed overnight at 37° C. in sterile RPMImedium containing gentamycin and antimycotic solution. Before challengeexperiments, 20 live infective L3s suspended in RPMI1640 mediumsupplemented with 15% heat inactivated fetal calf serum (FCS), wereintroduced into the micropore chambers and the opening was sealed withdental cement. Micropore chamber containing the L3s were then surgicallyimplanted into the peritoneal cavity of each mice under anaesthesia.Aseptic conditions were followed for the surgical procedures. After 48hours of implantation, animals were sacrificed and the chambers wererecovered from peritoneal cavity. Contents of each chamber were emptiedand larvae were examined microscopically for adherence of cells and forlarval death. Larval viability was determined microscopically at 100×.The percentage of protection was expressed as the number of deadparasites÷number of total parasites recovered×100.

Cytokine Analysis in Mice.

The percent of rBmHSP and rBmALT2 specific interferon-γ (IFN-γ) andinterleukin-4 (IL-4) secreting cells were determined in the spleen ofcontrol and vaccinated mice using an ELISPOT assay. Briefly, MILLIPOREMULTISCREEN HTS Filter plates were coated with monoclonal rat anti mouseIFN-γ or monoclonal rat anti-mouse IL-4 antibodies (BD Pharmigen, SanDiego, Calif.) at a concentration of 10 μg/ml in PBS buffer. Afterwashing the plates, non-specific sites were blocked by incubating thewells in complete RPMI with 10% fetal calf serum for one hour at roomtemperature. Approximately 3×10⁶ spleen cells suspended in completeRPMI1640 medium supplemented with 10% heat inactivated FBS were thenadded to each well. Cells were stimulated with rBmHSP or rBmALT2 (1μg/ml). Unstimulated cells served as controls. Forty-eight hours afterincubation at 37° C. in humidified 5% CO₂, plates were washed andfurther incubated for 1 hour at room temperature with 2 μg ofbiotinylated rat anti-mouse IFN-γ or biotinylated rat anti-mouse IL-4antibody (BD Pharmigen). After washing the plates,streptavidin-conjugated horseradish peroxidase (Thermo FisherScientific) was added (1:800) to each well and incubated at roomtemperature for one hour. Plates were washed and color developed usingDAB substrate (Thermo Fisher Scientific). Total numbers of spots werecounted under a dissection microscope.

Statistical Analysis.

Statistical analysis was performed using XL STAT software v.7.5.2(Kovach Computing Services, Anglesey, UK). Statistical significancebetween comparable groups was estimated using appropriate non-parametrictests, with the level of significance set at p<0.05.

Antibody Responses in Mice.

It was first determined whether the multivalent immunogenic compositioncould elicit significant antibodies against each of the antigeniccomponents. Previous studies have shown that mice similarly vaccinatedwith B. malayi antigens elicited significant host protective IgGantibodies (Veerapathran, et al. (2009) supra). Therefore, IgG antibodytiters were analyzed. The results of this analysis indicated that themonovalent immunization with Bmhsp+rBmHSP and Bmalt2+rBmALT2 elicitedsignificant (p<0.005) titers of anti-BmHSP and anti-BmALT2 IgGantibodies (FIG. 1). The multivalent immunogenic composition alsoelicited significant IgG antibody titers. Following multivalentimmunogenic composition, the mice produced IgG antibodies against bothBmHSP and BmALT2 equally, suggesting that the antigens do not interfereor compete for dominance. An interesting finding was that themultivalent immunogenic composition elicited 1.5- to 1.75-fold higher(p<0.005) titers of IgG antibodies compared to the monovalent vaccine(FIG. 1). These finding indicated that the two antigens in themultivalent formulation can act synergistically by increasing thevaccine-induced antibody responses against each antigen in thevaccinated mice. The findings also indicated that combining these twoantigens in the vaccine formulation has a great advantage. Given therobust IgG antibody responses induced following vaccination, it is alsopossible that the concentration of the component antigens in themultivalent preparation can be reduced.

Multivalent Immunogenic Composition Induces Significant Protection inMice.

The results herein showed that significant IgG antibodies were elicitedfollowing vaccination with monovalent and multivalent immunogeniccomposition preparations. To test if the immune responses elicitedfollowing vaccination were protective, vaccinated animals werechallenged with live third stage infective larvae (L3) of B. malayi.Since the parasites do not reach maturity in these animals, a betterrecovery of worms is obtained if the parasites are surgically implantedinto the animals. A standard micropore chamber challenge method(Abraham, et al. (1989) supra). These studies showed that close to 61%protection could be achieved in mice immunized with a monovalent vaccine(Table 8). This was highly significant (p<0.001) compared to negativecontrols. This finding also showed that rBmHSP and rBmALT2 are of use invaccines for lymphatic filariasis. Challenge experiments in miceimmunized with multivalent immunogenic composition showed thatsignificantly (p<0.005) higher protection could be achieved compared tomonovalent vaccination (Table 8). These findings also clearly correlatedwith the higher IgG antibody titer in these animals and support theabove finding that rBmALT2 and rBmHSP can synergistically enhance theprotective immune responses in vaccinated animals when given as a primeboost regimen (Table 8).

TABLE 8 Percent Vaccination regimen Larval Death^(a) Groups Bmhsp DNAprime and rBmHSP 61 ± 4.24 Monovalent protein boost Bmalt2 DNA prime andrBmALT2 76 ± 8.21 Monovalent protein boost Bmhsp + Bmalt2 prime andrBmHSP 90 ± 7.53 Multivalent and rBmALT2 protein boost pVAX plus alumcontrol  22 ± 10.41 Control ^(a)Values are mean + SD. N = 5. Data isfrom one of two similar experiments showing comparable results.

To further demonstrate efficacy, mice were immunized with variousprime-boost combinations. As shown in FIG. 3, 100% protection can beachieved in mice following immunization with HAT hybrid protein or afterprime boost immunization with HAT hybrid DNA and HAT hybrid protein.

Cytokine Responses.

The immunological characteristics of the protective responses invaccinated mice were determined by evaluating the secreted cytokineresponses of spleen cells in response to the vaccine antigens. Whenspleen cells were stimulated with rBmHSP or rBmALT there was significantantigen-specific proliferation of spleen cells suggesting a strongrecall cellular response to the antigens. To identify the cytokineprofile of these antigen-responding cells, the IFN-γ and IL-4 secretingcells were counted using an ELISPOT assay. Results from these studiesshowed that spleen cells from mice vaccinated with multivalentimmunogenic composition were predominantly secreting IL-4 (FIG. 2A). Thenumbers of IFN-γ secreting cells were very low (FIG. 2B). Overall, thesefindings indicated that vaccine-induced protection was largely mediatedby Th2 type responses.

Example 3: BmVal-1+BmALT2 Multivalent Immunogenic Composition

Sera. Sera samples used in this study were from archived samples storedat the Mahatma Gandhi Institute of Medical Sciences, Sevagram, India.These samples were collected as part of epidemiological surveys in andaround Wardha, an area endemic for lymphatic filariasis.

No demographic data was available to this study except that the serasamples were classified into microfilaremic (MF), chronic pathology (CP)or Endemic normals (EN) based on the detection of circulating parasites,parasite antigens or by evaluating clinical symptoms of lymphaticfilariasis. Circulating microfilariae were detected in the blood ofsubjects according to known methods (Haslbeck, et al. (2005) Nat.Struct. Mol. Biol. 12:842-846; Yoo, et al. (2005) Biotechnol. Lett.27:443-448). The presence of circulating antigen was detected using anOg4C3 kit and a WbSXP-based enzyme-linked immunosorbent assay (ELISA).Subjects with no circulating antigen or microfilariae were classified asEN, whereas subjects with circulating microfilariae and/or circulatingantigen, as detected by ELISA, were considered as MF. Subjects showinglymphedema and other visible clinical symptoms of filariasis weregrouped into CP. Control non-endemic normal (NEN) sera were collected atthe University of Illinois Clinic at Rockford, Ill.

Parasites.

Brugia malayi L3s were obtained from the NIAID/NIH Filariasis ResearchReagent Resource Center (FR3) at the University of Georgia, Athens, Ga.

Construction of Monovalent and Multivalent DNA Vaccines.

To prepare monovalent vaccine, codon optimized Bmval-1 or Bmalt-2 geneswere cloned into the eukaryotic expression vector pVAX1 (Invitrogen,Carlsbad, Calif.) using insert-specific primers (Yoo, et al. (2005)supra; Huang, et al. (2005) Immunol. Lett. 101:71-80). To preparemultivalent immunogenic composition, codon-optimized Bmval-1 gene wasfirst cloned into pVAX1 vector with no stop codon using alreadypublished primer sequences with a PstI site. Codon-optimized Bmalt-2gene was then inserted into this clone using gene-specific primers. PCRparameters for all the constructs were: 94° C. denaturation for 30seconds, 50° C. primer annealing for 30 seconds, 72° C. primer extensionfor 30 seconds for 30 cycles; and a final extension of 5 minutes wasperformed at 72° C. Insert DNA was sequenced to ensure authenticity ofthe cloned nucleotide sequence on both strands. Plasmids were maintainedand propagated in E. coli TOP10F′ cells. Plasmids were purified usingendotoxin-free plasmid extraction kit (Qiagen, Valencia, Calif.). DNAwas analyzed by agarose gel electrophoresis and quantified in aspectrophotometer (OD 260/280, ratio >1.8).

Expression and Purification of Recombinant Proteins.

Recombinant BmVAL-1 and rBmALT2 were expressed in pRSET-A vector andpurified using an immobilized cobalt metal affinity columnchromatography according to published methods (Norimine, et al. (2004)Infect. Immun. 72:1096-1106; Shinnick, et al. (1988) Infect. Immun.56:446-451). Endotoxin in the recombinant preparations were removed bypassing the recombinant proteins through polymyxin B affinity columns(Thermo Fisher Scientific, Rockford, Ill.) and the levels of endotoxinin the final preparations were determined using an E-TOXATE kit (Sigma,St Louis, Mo.) as per manufacturer's instructions. Endotoxin levels inthe final preparations (0.005 EU/ml) were below detection limits inthese recombinant protein preparations.

Immunoreactivity of Human Sera.

To determine if the human sera samples carried antibodies againstBmVAL-1 or BmALT2, an ELISA was performed (Haslbeck, et al. (2005)supra; Yoo, et al. (2005) supra). For isotype-specific ELISA, alkalinephosphatase-conjugated goat anti-human IgG1, anti-human IgG2, anti-humanIgG3, and anti-human IgG4 antibodies (Sigma) were used as the secondaryantibodies.

Immunization Protocol for Mice and Jirds.

Six-week old male Balb/c mice and 35-40 gm outbred male mongoliangerbils (jirds) purchased from Charles River Laboratories (Wilmington,Mass.) were used in these experiments. Animals were treated as per theguidelines in the Guide for the Care and Use of Laboratory Animals. Twodifferent animal models were used because B. malayi parasite does notmature into adults in mouse, so vaccine-induced protection against theL3 stages can be evaluated in the mouse model. In addition, significantimmunological parameters can be measured in mice. Conversely, B. malayiparasite develops into mature adult worms in jirds. Therefore,vaccine-induced protection can be evaluated against adult wormestablishment in jirds.

Three sets of experiments were performed: (1) monovalent BmVAL-1vaccination, (2) monovalent BmALT2 vaccination and (3) multivalentmVAL-1/BmALT2 vaccination. Each experimental set had four groups (a) DNAprime plus DNA boost (homologous), (b) protein prime plus protein boost(homologous), (c) DNA prime plus protein boost (heterologous) and pVAXplus alum controls. Each group included ten (10) animals each. Allanimals were immunized subcutaneously with codon-optimized DNA (100 μg)in 50 μl volume or with recombinant protein (150 μg) plus alum in 50 μlvolume. Control group received 100 μg of pVAX1 blank vector or 50 μl ofalum. Blood samples were collected at frequent intervals, sera separatedand stored at −80° C. The protocol used for immunizing mice and jirdswas as follows. Animals were prebled and given a first dose on day 0. Asecond dose was administered on day 14 and subsequently bled. Third andfourth doses were administered on days 28 and 42, respectively, and theanimals were subsequently bled. Mice were challenged on day 56 andprotection was determined on day 58. Jirds were challenged on day 60 andprotection was determined on day 155.

Protection Studies in Mice.

Challenge studies were conducted in mice by surgically implanting twentylive, infective B. malayi L3s into the peritoneal cavity in a microporechamber (Veerapathran, et al. (2009) supra; Abraham, et al. (1988)supra). Aseptic conditions were followed for the surgical procedures.Forty-eight hours after implantation, chambers were recovered from theperitoneal cavity and viability of the larvae was determined under alight microscope. The percentage of protection was expressed as thenumber of dead parasites+number of total parasites recovered×100.

Splenocyte Proliferation and Cytokine Assays.

Single-cell suspension of spleen cells (0.5×10⁶ cells per well suspendedin 200 μl media) were prepared from each mouse and cultured intriplicate wells with either (1) 1 μg/ml rBmVAL-1, (2) 1 μg/ml rBmALT2,(3) 1 μg/ml rBmVAL-1+BmALT2, (4) a nonspecific recombinant protein (1μg/ml of Schistosoma mansoni G-binding protein) or (5) were leftunstimulated in the media. All cells were incubated for 3 days at 37° C.with 5% CO₂. After 3 days, ³H-Thymidine (0.5 μCi per well, AmershamBiosciences) was added to each well and further incubated. Cells wereharvested 16 hours later and ³H-thymidine uptake was measured in aliquid scintillation counter and expressed as stimulation index(SI)=(counts per minute of stimulated cultures counts per minute ofunstimulated cultures). Cell culture supernatants collected from thespleen cultures were assayed for IFN-γ, IL-4, IL-5 and IL-10 using anELISA kit purchased from eBioscience Inc. (San Diego, Calif.).

BmVAL-1 and BmALT2 Specific IgG Antibodies in the Sera of ImmunizedMice.

Titer of anti-BmVAL-1- and anti-BmALT2-specific antibodies wasdetermined in the sera of immunized mice using an ELISA (Veerapathran,et al. (2009) supra; Gnanasekar, et al. (2004) Infect. Immun.72:4707-15). Pre-immune sera served as controls. HRP-conjugated goatanti-mouse IgG was used as the secondary antibody (Thermo FisherScientific) for mouse assays. OPD (Sigma) was used as the substrate andoptical density (OD) was measured at 405 nm.

Anti-BmVAL-1- and anti-BmALT2-specific IgG1, IgG2a, IgG2b, IgG3 and IgG4antibodies were determined in the sera of mouse using a mouse antibodyisotyping kit purchased from Thermo Fisher Scientific. All ELISAs wereperformed as per the manufacturer's recommendation and absorbance wasread at 405 nm. Respective HRP-labeled goat anti-IgG isotype antibodywas used as the secondary antibodies and color was developed using OPDsubstrate.

Challenge Studies in Jirds.

Jirds were challenged with 100 B. malayi L3s and worm establishment wasdetermined on day 95 after challenge according to established methods(Weil, et al. (1992) supra). Jirds are permissive hosts for B. malayiand the worms mature into adult males and females in about 75 days.Presence of mature worms in the control group of jirds was confirmed bydemonstrating microfilariae in their blood on day 80 after challenge.Percent reduction in the worm establishment was calculated using theformula: average number of worms recovered from control worms−averagenumber of worms recovered from vaccinated animals/average number ofworms recovered from control animals×100.

Statistical Analysis.

Statistical analysis was performed using SIGMASTAT program (JandelScientific, San Rafel, Calif.) and STATVIEW (SAS Institute, Cary, N.C.)software. Wilcoxon signed rank test was used to compare paired data;comparison between the groups was performed using the Mann-Whitney Utest. p value of p<0.05 was considered statistically significant.

EN individuals Carry High Titer of Antibodies Against BmVAL-1 andBmALT2.

Significant anti-BmVAL-1 and anti-BmALT2 IgG antibodies were present inthe sera of EN subjects compared to MF subjects (p<0.01) and CP subjects(p<0.005). NEN subjects did not carry IgG antibodies against either ofthe antigens. Subsequent analysis of the IgG isotype of antibodies inthe sera of EN subjects showed that anti-BmVAL-1 and anti-BmALT2antibodies were predominantly of IgG1 and IgG3 isotypes.

High Titer of Antibody Responses in the Sera of Immunized Mice.

It has been shown that mice vaccinated with B. malayi antigens elicitsignificant host protective IgG antibodies. Therefore, IgG antibodytiters in the sera of immunized mice were determined. Monovalentimmunization with BmVal-1 and monovalent immunization with BmAlt2 bothelicited significant (p<0.005) titers of anti-BmVAL-1 and anti-BmALT2IgG antibodies in the sera of mice. Compared to controls, the primeboost immunized group gave the maximum titer of antibodies followed byprotein immunized and DNA immunized groups. Immunization with themultivalent immunogenic formulation (BmVAL-1+BmALT2) also elicitedsignificant IgG antibody titers against both rBmVAL-1 and rBmALT2 andthe titers were comparable, indicating that the antigens do notinterfere with each other or compete for dominance. An interestingfinding was that the multivalent immunogenic elicited significantlyhigher (p<0.001) titer of IgG antibodies in mice compared to any of themonovalent vaccines. These finding indicated that the two antigens inthe multivalent formulation synergistically increased thevaccine-induced antibody responses.

Overall, protein vaccination elicited higher titer of IgG antibodiescompared to DNA vaccines, indicating that protein vaccinations werehighly immunogenic. Another observation was that a heterologous primeboost approach gave a higher seroconversion than homologous prime boostapproach. Thus, overall heterologous prime boost approach appeared tostimulate the highest titer of antibodies.

IgG antibody subset analysis showed that BmVAL-1 vaccination elicitedprimarily IgG1 and IgG2a isotype of antibodies, whereas, BmALT2vaccination induced IgG1, IgG2a and IgG3 isotype of antigen-specificantibody responses. Antigen-specific IgG4 antibody responses were notevident. The prime boost approach significantly amplified the IgGisotype responses. Following multivalent vaccination regimen IgG1, IgG2aand IgG3 subset of antigen specific antibodies were present in the seraof mouse.

Antigen-Specific Responses in the Spleen of Mice.

Spleen cells from immunized mice stimulated with either rBmVAL-1 orrBmALT2 proliferated significantly (SI 10.8±1.1 and SI 14.6±1.2,respectively) compared to the media control (SI 2.1±0.9). Spleen cellsfrom mice immunized with the multivalent construct responded to bothrBmVAL-1 (SI 18.9±2.6) and rBmALT2 (SI 23.5±3.1), indicating that astrong recall cellular response was generated to both BmVAL-1 and BmALT2following vaccination with the multivalent construct.

Cytokine Analysis from Proliferated Culture Supernatants.

To identify the cytokine profile of the antigen-responding cells, theculture supernatant of mouse spleen cells stimulated with respectiveantigen (rBmVAL-1 or rBmALT2) was collected and the level of IFN-γ,IL-4, IL-5 and IL-10 was measured. These results showed that significantlevels of IL-5 and IFN-γ were secreted by the spleen cells in responseto rBmVAL-1. Spleen cells stimulated with rBmALT2 predominantly secretedIL-4 and IL-5.

Multivalent Immunogenic Composition Induces Significant Protection inMice and Jirds.

The results herein indicated that significant IgG antibodies wereelicited following vaccination with monovalent and multivalentimmunogenic preparations. To test if the immune responses elicitedfollowing vaccination were protective, vaccinated animals werechallenged with live, third stage infective larvae (L3) of B. malayi.Since the parasites do not reach to maturity in mice, a standardmicropore chamber challenge method was used (Gnanasekar, et al. (2004)supra). These studies showed that 39% to 74% protection was achieved inmice following immunization with monovalent vaccine (Table 9).

TABLE 9 Mean ± SD Percent Vaccination Group Live L3s ProtectionpVAXBmVAL-1 DNA monovalent 12.2 ± 4.5   39.0 ± 1.7%** homologousrBmVAL-1 protein monovalent 10.4 ± 3.1  48.0 ± 2.1%* homologouspVAXBmVAL-1 DNA plus rBmVAL-1 9.2 ± 2.2 54.0 ± 3.1%* monovalentheterologous pVAXBmALT2 DNA monovalent 9.8 ± 2.1 51.0 ± 2.5%* homologousrBmALT2 protein monovalent 7.0 ± 1.1 65.0 ± 4.2%* homologous pVAXBmALT2DNA plus rBmALT2 5.1 ± 0.5 74.5 ± 3.1%* monovalent heterologouspVAXBmVAL-1/ALT2 DNA 8.6 ± 0.1 57.0 ± 2.2%* multivalent homologousrBmVAL-1/rBmALT2 protein 5.2 ± 1.1 74.0 ± 3.3%* multivalent homologouspVAXBmVAL-1/BmALT2 DNA plus 4.4 ± 0.4 82.0 ± 2.2%* rBmVAL-1/rBmALT2multivalent heterologous pVAX + Alum control 20 ± 0  0% Significance, *p< 0.01, **p < 0.05 compared to control.

Protein vaccination gave better results than DNA vaccination. The primeboost regimen gave the best results overall. Vaccination with BmALT2gave higher percent of protection compared to BmVAL-1. Similarly,multivalent vaccination regimen gave the 57% to 82% protection comparedto the monovalent vaccination regimen. These finding indicated thatBmVAL-1 and BmALT2 synergistically enhance the protective immuneresponses in vaccinated animals when given as a multivalent immunogeniccomposition.

Analysis of the thick blood smear prepared from the control group ofjirds on day 80 after challenge showed that all five jirds were positivefor microfilaria, whereas, microfilaria were not detected in theperipheral blood of vaccinated jirds. Fifteen (15) days later theanimals were sacrificed and the male and female worms in the peritoneal,pelvic and pleural cavities were counted and the results betweencontrols and vaccinated groups were compared (Table 10). Findings fromvaccination of jirds also confirmed that the multivalent prime boostregimen gave the highest rate of protection. No female worms wererecovered from the multivalent vaccinated animals.

TABLE 10 Vaccination Group Percent Production pVAXBmVAL-1 DNA monovalent 50 ± 3.7% homologous rBmVAL-1 protein monovalent 40.0 ± 3.1% homologouspVAXBmVAL-1 DNA plus rBmVAL-1 52.4 ± 2.5% monovalent heterologouspVAXBmALT2 DNA monovalent 58.3 ± 2.1% homologous rBmALT2 proteinmonovalent 72.0 ± 5.5% homologous pVAXBmALT2 DNA plus rBmALT2 78.5 ±3.2% monovalent heterologous pVAXBmVAL-1/ALT2 DNA 77.1 ± 2.0%multivalent homologous rBmVAL-1/rBmALT2 protein 79.9 ± 3.5% multivalenthomologous pVAXBmVAL-1/BmALT2 DNA plus 85.0 ± 1.4% rBmVAL-1/rBmALT2multivalent heterologous pVAX + Alum control 0 Significance, p < 0.01compared to control.

Example 4: BmHSP+BmALT2+BmTSP Multivalent Immunogenic Composition

Parasites. Brugia malayi L3s were obtained from the NIAID/NIH FilariasisResearch Reagent Resource Center (FR3) at the University of Georgia,Athens, Ga.

Construction of pVAX Bmhsp+Bmalt2+Bmtsp DNA vaccine. The codon-optimizedDNA sequence coding for Bmhsp was amplified with the forward primer5′-CGC GGA TCC ACC GTG ATC CAT TGT CG-3′ (SEQ ID NO:25) containing BamHIrestriction site and the reverse primer 5′-AAC TGC AGC TGT TTT CCA TTTCCA TTC-3′ (SEQ ID NO:26) containing PstI restriction site without thestop codon and cloned into pVAX vector. The resulting plasmid wasdesignated as pVAX Bmhsp*. Codon-optimized Bmalt2 gene was amplifiedwith the forward primer 5′-AAC TGC AGA TGG GTA ACA AGC TCC TCA TCG-3′(SEQ ID NO:27) and the reverse primer without the stop codon 5′-CGC GAATTC GGC GCA CTG CCA ACC TGC-3′ (SEQ ID NO:28). Underlined sequencesindicate PstI and EcoRI restriction sites in the forward and reverseprimers, respectively. The amplified Bmalt2 DNA insert was thensubcloned into pVAX Bmhsp* plasmid at the PstI and EcoRI restrictionsites, resulting in pVAX Bmhsp+Bmalt2* plasmid. To clone the finalproduct of pVAXBmhsp+Bmalt2+Bmtsp plasmid, the gene sequence encodingBmtsp ECL domain alone was amplified with the forward primer 5′-CGC GAATTC ACC ATG GTC CTG GAG-3′ (SEQ ID NO:29) containing EcoRI restrictionsite and the reverse primer with stop codon 5′-GCT CTA GAT CAG TCC TTCTGG CTA G-3′ (SEQ ID NO:30) containing XbaI restriction site and clonedinto pVAX Bmhsp+Bmalt2* plasmid. Bivalent constructs of HSP+TSP, TSP+ALTand HSP+ALT were also constructed with their respective primers.

Construction of pRSETA Bmhsp+Bmalt2+Bmtsp, a Multivalent Fusion Protein.

The Bmhsp+Bmalt2+Bmtsp fusion protein was constructed in the same manneras above. The primer sequences of HSP, ALT2 and TSP were as follows.Bmhsp, forward primer, 5′-CGG GAT CCA TGG AAG AAA AGG TAG TG-3′ (SEQ IDNO:31) containing BamHI and reverse primer, 5′-CCC TCG AGT GCT TTC TTTTTG GCA GC-3′ (SEQ ID NO:32) containing XhoI. Bmalt2, forward primer,5′-CCC TCG AG A TGA ATA AAC TTT TAA TAG CAT-3′ (SEQ ID NO:33) containingXhoI or 5′-AAC TGC AGA TGG GTA ACA AGC TCC TCA TCG-3′ (SEQ ID NO:27) andreverse primer, 5′-GGG TAC CCG CGC ATT GCC AAC CC-3′ (SEQ ID NO:34)containing KpnI. Bmtsp, forward primer, 5′-GGG GTA CCC CGG CAA GGA TCAATT TAA AA-3′ (SEQ ID NO:35) containing KpnI and reverse primer, 5′-CGGAAT TCT CAA TCT TTT TGA GAT GAA T-3′ (SEQ ID NO:36) containing EcoRIwere used to amplify the Bmtsp fragment of SEQ ID NO:77. Primers werealso designed to amplify a Tetraspanin Large Extracellular Loop (LEL)fragment of SEQ ID NO:63. Bivalent constructs (HA, HT and TA) were alsocloned individually into a pRSETA vector.

Immunization of Animals.

Six-week-old Balb/C mice were immunized with 100 μg of DNA intradermally(i.d.) as DNA vaccine or with 15 μg of recombinant proteinsubcutaneously (s.c.) as protein vaccine or with two doses of DNA andtwo doses of protein as prime-boost vaccine. Mice were randomly dividedinto 15 groups with 5 mice per group. Animals from groups 1-3 wereimmunized with HSP+ALT2 (HA). Groups 4-6 were immunized with HSP+TSP(HT), and 7-9 were immunized with TSP+ALT2 (TA). Mice from groups 10-12were immunized with the multivalent immunogenic composition HSP+ALT2+TSP(HAT). Control group of animals received pVAX vector and/or alum(Infectious Disease Research Institute (IDRI)). This experiment wasrepeated twice with all the groups.

Analysis of Antibody Response in Immunized Animals.

IgG antibody levels in the sera of immunized and control groups ofanimals against all the three proteins were determined using an indirectELISA (Anandharaman, et al. (2009) supra). Briefly, wells of a 96-wellmicrotiter plate were coated with recombinant proteins (rHSP, rALT2 orrTSP; 1 μg/ml) in carbonate buffer, pH 9.6, overnight at 4° C. andblocked with 3% BSA for 1 hour at 37° C. Sera samples were added to thewells and the plates were incubated overnight at 4° C. After washing,HRP-labeled mouse anti-human IgG was added (1:5000) and incubatedfurther for 1 hour at 37° C. The color was developed with OPD(o-phenylene diamine) substrate (Sigma Aldrich, USA). Absorbance wasmeasured at 450 nm in a microplate reader (BIO-RAD, Hercules, Calif.).

Immunoblot analysis was also performed with the immunized mice sera.Sera samples from the mice immunized with the recombinant proteins(rHAT, rALT2, rTSP or rBmHAT) were used for the immunoblot study. Thecolor of the blot was developed with the diaminobenzidine (DAB)substrate.

ADCC Assay.

To evaluate the protection efficacy of the antigen combinations, invitro ADCC was performed with the sera from the mice immunized withbivalent and trivalent vaccine constructs. The in vitro ADCC assay wasperformed according to known methods (Chandrasekhar, et al. (1990)supra). Briefly, Peritoneal Exudates Cells (PEC) were collected fromnormal Balb/c mice by washing the peritoneal cavity with sterile RPMI1640 media. The cells were washed and suspended in RPMI 1640 mediumsupplemented with 10% Fetal Calf Serum (FCS). Ten L₃ of B. malayi wereadded to 2×10⁵ peritoneal exudates cells (PEC)/well in 96-well cultureplates (Thermo Fisher Scientifics, USA.), 50 μl of immunized mice seraand 50 μl of RPMI 1640 media were added to the wells in triplicates andincubated for 48 hours in 5% CO₂ at 37° C. Larval viability wasdetermined microscopically after 48 hours of incubation. Larvae thatwere limpid, damaged and with the clumps of cells adhered to it werecounted as dead. ADCC was estimated as the percent larval deathcalculated using the formula: Number of Dead larvae÷Total number oflarvae×100.

Depletion of IgG Antibodies from the Sera Samples.

Sera from mice immunized with multivalent immunogenic composition wasdepleted of recombinant antigen specific IgG antibodies using cobaltIMAC resin coupled with his-tagged recombinant antigens (Anandharaman,et al (2009) supra). Briefly, 1 mg of his-tagged recombinant protein(rHSP) was coupled to 2 ml bed volume of IMAC resin for 2 hours at 37°C. The cobalt column was washed with ten bed volumes of PBS (pH. 8) andincubated overnight at 4° C. with 200 μl of pooled sera from the miceimmunized with multivalent immunogenic composition. Supernatantcontaining the depleted sera was collected by centrifugation.Anti-HSP-depleted serum was incubated overnight at 4° C. inrALT2-coupled column. The supernatant containing anti-HSP- andanti-ALT2-depleted serum was collected and incubated in rTSP-coupledcolumn. Anti-HSP-, anti-ALT2-, and anti-TSP-depleted serum was collectedand used. Depletion of IgG antibodies against specific antigens wasconfirmed by ELISA as described above. Antibody-depleted sera were thenused in an ADCC assay.

Analysis of In Situ Cytotoxicity Against L3 Larvae in Immunized Mice(Micropore Chamber Technique).

The protective efficacy of vaccination was analyzed by challenging theimmunized animals with infective L3 using micropore chamber method(Abraham, et al. (1989) supra) Micropore chambers were assembled using14×2 mm PLEXIGLASS (acrylic) rings and 5.0 μm NUCLEOPORE polycarbonatemembranes (Millipore Corporations, Bedford, Mass.). After 48 hours ofimplantation, animals were sacrificed and the chambers were recoveredfrom peritoneal cavity. Contents of each chamber were examinedmicroscopically for cell adherence and death of infective L3. Theparasite was considered dead if it was not motile and limpid, and hadseveral adherent cells on the surface. The percentage protection wascalculated using the formula: number of dead parasites÷number ofrecovered parasites×100. This experiment was repeated twice with fiveanimals in each group.

Splenocyte Proliferation.

Vaccinated and control mice were sacrificed on day 60 and the spleenswere removed aseptically. Single-cell suspensions were prepared in RPMI1640 medium supplemented with 10% heat-inactivated FCS, passed through aNYLON (aliphatic polyamide) mesh (BD Biosciences, Bedford, USA). Afterdetermining the viability of cells using trypan blue dye exclusion,approximately 2×10⁶ cells per well in triplicates were plated in 96-wellculture plates (ThermoFisher, USA). The splenocytes were stimulated with1 μg/100 μl/well of recombinant proteins (rHSP, rALT2 or rTSP) or ConAor with medium alone (Unstimulated) for 72 hours at 37° C. in theatmosphere of 5% CO₂. Cell proliferation was determined using cellcounting kit (CCK-8) purchased from Dojindo Molecular Technologies, Inc.(Gaithersburg, Md.). Stimulation index of spleen cell proliferation wascalculated using the formula: Absorbance of stimulated cells÷Absorbanceof unstimulated cells. All cultures were taken in triplicates and theresults expressed as mean S.I.±SEM.

Real Time-PCR (RT-PCR). Cytokine levels in the mRNA of the spleen cellpellets were analyzed by real time-PCR. The spleen cells of vaccinatedand control group mice were cultured as above at a concentration of2×10⁶ cells/100 μl/well in 96-well plates and stimulated withrecombinant antigens (1 μg/ml). After 72 hours, cells were centrifuged(1000 rpm for 5 minutes) and total RNA was extracted from the cellpellets using TRIZOL (phenol, guanidinium and thiocyanate) reagent(Invitrogen) as per description of the manufacturer. Followed by RNAextraction, first-strand cDNA was synthesized by RT² First Strand Kit(SuperArray Bioscience Corporation, Frederick, Md.). PCR array analysiswas performed according to the manufacturer protocol with the RT²Real-Time™ SYBR Green (cyanine dye) PCR Master Mix. Aliquots from thismix were added to a 96-well plate, where each well containedpredispensed gene-specific primer sets. Relative quantification of thegenes of interest that expressed was measured in an Applied BioSystem7300 real-time PCR machine (Applied BioSystems, Foster City, Calif.).Cycling parameters were as follows: 95° C. for 10 minutes for activationof HOTSTART DNA polymerase, followed by 40 cycles of denaturation at 95°C. for 15 seconds and primer extension at 60° C. for 1 minute. RT-PCRdata array set was generated and analyzed using SABiosciences web-baseddata analysis system. Results were expressed in terms of fold change ofimmunized mice compared to control mice by normalizing the expression ofhousekeeping genes.

Cytokine Assay.

Splenocyte cell culture supernatants were collected after 72 hoursincubation stimulated with recombinant antigens (1 μg/ml) or with mediumalone. Secreted levels of IL-4 and IFN-γ cytokines in the culturesupernatants were determined using a sandwich ELISA kit purchased fromThermo Scientifics, USA. All concentrations were derived from standardcurves and data expressed in pg/ml.

Construction of cHAT Plasmid and Expression of Fusion Proteins.

Since the N-terminal region of HSP is involved in IL-10 binding, thisregion was deleted and cHAT recombinant protein was prepared as 37 KDaHis-tagged protein.

Construction of Recombinant Plasmids and Expression of Fusion Proteins.The full-length hsp, alt2 and tsp genes of B. malayi L3 stage wereconstructed with the expected size (850 bp). These fragments werefurther directionally cloned into the expression vectors pVAX1 andpRSETA with the specified restriction enzyme cutting sites. Results ofthe DNA sequence analysis confirmed gene insertion direction. rBmHAT wasexpressed as a 45 KDa His-tagged fusion protein, which was purified andanalyzed in SDS-PAGE. The results indicated that the fusion protein waspure without any contaminating proteins. The presence of antibodiesagainst all the three antigens was confirmed by immunoblot analysis.

Antibody Titer in the Immunized Mice Sera.

The mean peak antibody titer of the sera samples from the mice immunizedwith prime-boost or protein vaccine was significantly higher (p<0.001)compared to the DNA group. Sera collected from rBmHAT-immunized animalsshowed the maximum titer of 30,000 against rALT2 antigen, while theantibody titer against rHSP or rTSP antigen was in the range of18,000-20,000. Similarly, the mice immunized with the bivalent vaccineshowed the maximum titer of 30,000 against ALT2 antigen while anti-HSPand anti-TSP antibodies were in the range of 8,000-15,000.

Antibody-Dependent Cell-Mediated Cytotoxicity.

Antibody-mediated adherence and cytotoxicity of immune cells to B.malayi L3 larvae was observed after 48 hours of incubation of parasites,with the sera and normal immune cells. ADCC showed maximum cytotoxicityof approximately 90% (p<0.001) in the sera of mice immunized with rBmHATor rWbHA vaccine constructs (Table 11). Bivalent vaccine constructs ofrWbHT and rWbTA also gave better protection of 82% and 87%,respectively, which was significant compared to monovalent-vaccinatedand control animals (p<0.001). To evaluate the protection mediated bythe antibodies generated against HSP, ALT and TSP antigens, IgGantibodies were depleted from the immunized sera and used in ADCC.Depleted antibodies showed only 6% protection against L3.

TABLE 11 Groups % Cytotoxicity H + A   90 ± 2.4* H + T  82.30 ± 12.9*T + A 87.06 ± 9.8* H + A + T 88.69 ± 7.5* anti-HSP + anti-ALT + anti-5.55 ± 1.5 TSP antibodies depleted from HAT immunized sera Valuesrepresent mean ± SD of three wells. *Significant larval death (P <0.001) compared to other mice groups.

In Situ Protection Study.

Two weeks after the final immunization, the ability of the vaccinecandidates, to kill the filarial parasites in the immunized animals wasevaluated by in situ micropore chamber studies. The data was combinedfrom the two similar experiments and represented as mean count±SEM. Theanalysis of percentage reduction in worm burden compared with controlshowed that multivalent immunogenic composition (HAT) conferred themaximum protection of 100% and 94% for protein and prime-boost vaccine,which was very significant protection (Table 12) (P<0.0001) compared tocontrol groups (5%). Interestingly, the percentage worm reduction ofbivalent vaccines HA, TA and HT were 90%, 80% and 82%, respectively,which was also significantly high compared to the control. In the entirebivalent vaccine group, prime-boost vaccination was more protectivecompared to DNA and protein vaccination.

TABLE 12 Trial 1 Trial 2 Trial 3 DNA Vaccine Protein Vaccine Prime-BoostVaccine % % % Group Cytotoxicity Group Cytotoxicity Group CytotoxicitypVAX 5 ± 4.23  Alum 3 ± 4.23  pVAX + Alum 5.9 ± 4.23   H + A 81 ± 11.23*H + A 78 ± 11.23* H + A 90 ± 11.23* H + T 72 ± 12.03* H + T 69 ± 12.03*H + T 80 ± 12.03* T + A 74 ± 11.21* T + A 66 ± 11.21* T + A 82 ± 11.21*H + A + T 91 ± 11.92* H + A + T 100 ± 0*    H + A + T 94 ± 11.92* Valuesare mean ± SD. N = 5. Data is from one of two similar experimentsshowing comparable results. *Significant larval death (P < 0.001)compared to other mice groups.

Splenocyte Proliferation.

Spleen cells isolated from vaccinated and control animals werestimulated in vitro individually with rHSP, rALT2 or rTSP to analyze theprotein-specific T-cell proliferation in vaccinated animals. Miceimmunized with the prime-boost regimen in all the vaccine combinationsand HAT as protein vaccine gave the highest protection. Hence thesplenocytes were collected only from these animals analyzed for theimmune response. Splenocytes from bivalent- and trivalent-vaccinatedanimals stimulated with respective recombinant proteins showedsignificantly high (P<0.001) proliferation (mean S.I.=4.25-5.8) whencompared to monovalent and unstimulated controls. The proliferationindex of spleen cells immunized with the monovalent construct showedsignificant proliferation. The stimulation of cells was comparable tothe positive controls.

RT-PCR Array.

To determine the cellular immune responses to multivalent constructs inthe vaccinated mice, spleen cells collected from vaccinated and controlmice were cultured in the presence of respective recombinant proteinsand their proliferative responses and cytokine profiles were evaluated.Since the spleen cells from vaccinated animals were proliferatingsignificantly to recall response, levels of cytokine mRNA were measured.An RT-PCR cytokine gene array was performed on mRNA collected from thespleen cells stimulated with recombinant proteins. These results showedthat both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokine genes weresignificantly increased in vaccinated animals.

Cytokine Levels.

After identifying the presence of IFN-γ and IL-4 cytokine expression inthe mRNA isolated from the vaccinated spleen cells, the secretion ofsame cytokines in the supernatant was investigated. The data werenormalized with the unstimulated controls. Interestingly, the cytokineprofiles observed in the supernatant exhibited significantly higherlevels of IFN-γ showing a Th1-biased immune response. These resultsdemonstrated that recombinant proteins stimulated the production ofIFN-γ and induced a Th1-mediated protective response.

Example 5: Analysis of cHAT Vaccine in Various Adjuvant Formulations

Preparation of cHAT.

Previous studies showed that the N-terminal sequence of BmHSP12.6 canbind to human IL-receptor and trigger IL-10-mediated responses(Gnanasekar, et al. (2008) Mol. Biochem. Parasitol. 159(2):98-103).Since IL-10 is an immunosuppressive agent, the IL-10 receptor bindingsequences were deleted from HSP. The truncated sequence was referred toas cHSP. The cHSP was then used to replace the HSP gene and HSP proteinin the multivalent HAT hybrid vaccine. Thus, the resulting new vaccinewas called cHAT.

Protection Studies Using cHAT-Fusion Protein Vaccine in Mice.

Mice were immunized with four doses of cHAT fusion protein at two-weekintervals. One month after the final immunization, the ability of thevaccine candidates to kill the filarial parasites was evaluated by insitu micropore chamber studies. Results showed that when mice wereimmunized with cHAT fusion protein with alum as the adjuvant, thevaccine conferred 81% protection (Table 13) (P<0.0001) compared tocontrol groups (2%) that received only phosphate-buffered saline (PBS)and alum. Different adjuvants were then tested to see if changing theadjuvant would improve the protection ability of cHAT. Two additionaladjuvants were tested: alum containing a TLR4 agonist (purchased fromInfectious Disease Research Institute, Seattle, Wash.) and ALHYDROGEL(purchased from Sigma, St. Louis, Mo.). cHAT with no adjuvants remainedas a control. Results from these studies (Table 13) showed that 78%protection was achieved with alum plus TLR4 agonist and cHAT given inALHYDROGEL adjuvant gave 70% protection. An interesting finding in thesestudies was that cHAT without any adjuvant also gave 72% protectionindicating that the cHAT fusion protein vaccine could be administeredwithout any adjuvant and still obtain significant protection.

TABLE 13 % Larval Death Group (Mean ± SD) cHAT + Alum  81 ± 7.8 PBS +Alum Control 1.7 ± 1.3 cHAT + Alum with TLR4 agonist  78 ± 8.4 cHAT +ALHYDROGEL 70 ± 13 cHAT With No adjuvant 72 ± 12 Values are mean ± SD. N= 5. Data is from one of two similar experiments showing comparableresults. *Significant larval death (P < 0.001) compared to other micegroups.

Example 6: Homologues of HSP, ALT2 and TSP

Homologues of the vaccine antigens, HSP, ALT2 and Tetraspanin arepresent in O. volvulus and L. loa. Comparison of the nucleotide sequenceof HSP, ALT2 and Tetraspanin from O. volvulus and L. loa show that thereis significant sequence homology (>90%) between the proteins from allfilarial parasites. These findings indicate that the cHAT fusion proteinvaccine developed in Example 5 can be used as a vaccine against O.volvulus and L. loa.

As an example, O. volvulus tetraspanin was cloned from O. volvulus L3cDNA library and recombinant proteins were prepared. Sera sample frommice vaccinated with cHAT vaccine that gave the 81% protection in Table13 was used to probe the recombinant O. volvulus tetraspanin afterseparating the protein in a 12% SDS-PAGE gel. B. malayi tetraspanin wasused as a positive control. Results showed that the sera samplesignificantly reacted with O. volvulus tetraspanin thereby indicatingthat the cHAT vaccine developed in Example 5 is of use as a vaccineagainst O. volvulus.

Example 7: Multivalent Immunogenic Composition Against LymphaticFilariasis in Rhesus macaque Model

Parasites.

B. malayi infective third stage larvae (L3) were obtained from theNIAID/NIH Filariasis Research Reagent Resource Center (University ofGeorgia, Athens, Ga.).

Multivalent Fusion Protein rBmHAT.

The multivalent fusion protein rBmHAT expressed in Escherichia coli BL21(pLysS), was purified and endotoxin removed by Pierce High CapacityEndotoxin removal resin column (Thermo Fisher Scientific, Rockford,Ill.) as described herein.

Immunizations of rBmHAT.

Five macaques each received 200 μg of rBmHAT vaccine mixed with 100 μgof AL007 alum (IDRI, Seattle, Wash.) under ABSL-2 conditions. Five (5)macaques that received alum (AL007) only remained as controls. Eachanimal was anesthetized with ketamine/xylazine and the vaccine wasadministered intramuscularly in each thigh (one injection site per thighper vaccination). Animals were immunized at 4-week intervals on days 0,28 and 56. Intramuscular route is commonly used for clinical vaccinetrials and hence the same procedure was followed for macaques. Theinjection sites were monitored daily for signs of fever, any adversereactions (redness, swelling, etc.) for up to 7 days post immunization.

B. malayi L3 Challenge.

On day 84, one month after the final dose of vaccine, macaques wereanesthetized with ketamine HCl and challenged subcutaneously with400-500 B. malayi L3. To facilitate the production of the relativelylarge number of L3 (500 L3/animal) required for challenging 10 immunizedmacaques, the animals were divided into 2 subgroups within each group.The subgroups were challenged one week apart. Before challenge, B.malayi L3 were counted and examined for viability under a microscope.Only viable parasites were used for challenge.

Monitoring of Each Animal After Challenge.

All animals were monitored daily for clinical signs after the challenge.Behavioral observations were similarly conducted during the entirepost-challenge period. Clinical monitoring included serum chemistry,hematology, complete blood count (CBC) analysis (IDEXX) and CD4+/CD8+ Tcell flow cytometry analysis. Body weights, body condition, lymphoedemaand lymph node measurements were also recorded each time the animal wassedated for procedures (like immunizations, challenge, and bloodcollections).

Sample Collection.

Blood samples and peripheral blood mononuclear cells (PBMC) werecollected. Whole blood was collected into BD VACUTAINER SST tubesaccording to manufacturer's instructions. Heparinized blood (1 ml) wascollected from the femoral vein of each animal during the immunizationperiod and from the saphenous vein during the challenge period. Theshift in blood collection site was to eliminate any potentialinterference with the inguinal lymph node measurements or assessments ofedema. Blood samples were obtained at multiple time points during theentire follow-up period.

Isolation of PBMC.

The blood pellets after plasma separation was diluted inphosphate-buffered saline (PBS; 1:2) and subjected to gradient densitycentrifugation for 30 minutes at 2200 rpm using a 90% HISTOPAQUEseparation solution (Sigma, St. Louis, Mo.). The opaque interfacecontaining mononuclear cells was collected, washed three times in PBS bycentrifugation at 800 rpm. PBMC were enumerated using Trypan blue dyeexclusion method and resuspended in RPMI 1640 medium containing 10% FBS(100 U/ml Penicillin/Streptomycin, and 2 mM L-glutamine). PBMC collectedbefore the challenge was analyzed for T cell proliferation and IFN-γsecretions. PBMC collected after the challenge experiments were testedfor T cell proliferation and ELISPOT assays. Proliferation assay wasperformed with PBMC isolated on the same day of blood collection. PBMCsuspended in RPMI media with 10% FBS were used for ADCC assay and forcytokines analysis.

T Cell Proliferation and Flow Cytometry.

Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay wasused for assessment of antigen-specific proliferation within the T cellpopulation (Parish, et al. (2009) Curr. Protoc. Immunol. Chapter 4: Unit49). A 5 mM CFSE stock solution (Invitrogen, Grand Island, N.Y.) wasprepared according to manufacturer's instructions. PBMC collected fourweeks after the final immunization were gently resuspended at 10⁷cells/ml in 5 μM CFSE and incubated in the dark at 37° C. for 15minutes. Cells were centrifuged and washed with RPMI containing 10% FBS(100 U/ml Penicillin/Streptomycin, and 2 mM L-glutamine) and incubatedfor an additional 30 minutes at 37° C. Cells were then washed,resuspended in RPMI containing FBS, plated in a 24-well plate at 2×10⁶cells/ml per well and incubated overnight at 37° C. The medium (500 μl)was removed the following day and cells were stimulated with 1 μg/mL ofrBmHAT. Samples incubated only with RPMI medium served as negativecontrols. As a positive control for each animal, cells were stimulatedwith phytohemagglutinin (PHA). Cells were cultured and harvested after 5days of stimulation. Following a washing step with PBS/0.2% FBS, cellswere surface stained with an antibody cocktail of CD3-APC-Cy, CD4-PE andCD8-PerCP and incubated for 20 minutes at room temperature. After anadditional washing step with PBS/0.2% FBS the cells were acquired on BDFACS CANTO II flow cytometer (BD, San Jose, Calif.) and analyzed on a BDFACS DIVA Software v6.1.2. At least 50,000 events within the livelymphocyte gate were acquired.

Cell Counts, Serum Chemistry and Complete Blood Count (CBC) Analysis.

CBC, serum chemistries and eosinophil counts were analyzed usingcommercial automated hematology and serum chemistry analyzers by IDEXX.Samples collected prior to the initiation of the study served as anormal reference baseline for each animal.

Measurement of Secreted Levels of IFN-γ.

PBMC (1×10⁶ cells) were stimulated in vitro with 1 μg/ml of rBmHAT for 5days at 37° C. Following stimulation, the supernatants were harvestedand assayed for secreted levels of IFN-γ using an ELISA kit (Mabtech AB,Ashburn, Va.) according to manufacturer's instructions.

ELISPOT Assay.

An ELISPOT assay was performed to determine the antigen-specific IFN-γand IL-10 secreting cells in the PBMC of vaccinated and controlmacaques. A monkey ELISPOT kit purchased from U-Cytech biosciences(Yalelaan, The Netherlands) was used to determine the spot forming unitsas per the manufacturer's instruction. PBMC collected 20 weeks postchallenge were plated in 96-well plates at 1×10⁶ cells/ml and werestimulated with 100 ng/well of B. malayi adult soluble antigen (BmA) for24 hours at 37° C. and 5% CO₂. Wells of ELISPOT plates were coated with100 μl/well of capture antibodies (anti-IL-10 or anti-IFN-γ) diluted insterile coating buffer and incubated overnight at 4° C. Plates werewashed 2 times with sterile coating buffer. After blocking the plateswith 200 μl/well of blocking buffer for 1 hour at room temperature, PBMCthat were already stimulated with BmA antigens or only media (negativecontrol) were added to the wells of the ELISPOT plates at 100 μl/welland incubated for 24 hours at 37° C. and 5% CO₂. All the cells wereremoved from the plates and the membrane was washed 3 times with sterilePBS. Following wash, 100 μl of detection antibodies were added to eachwell and incubated at room temperature for 2 hours. After washing theplate 4 times with wash buffer, avidin-HRP reagent was added (100μl/well) and incubated for 45 minutes at room temperature. After a finalwash with PBS, freshly prepared 3-amino-9-ethylcarbazole (AEC) substratesolution was added (100 μl/well) and monitored for the development ofspots at room temperature for 10-60 minutes. The substrate reaction wasstopped by washing wells 3 times with 200 μl/well ultrapure water. Theplates were air dried. Spots were counted using a dissecting microscope.The plates were stored in the dark prior to reading. Antigen-specificresponses were determined by subtracting the number of spots in thenegative control wells from the wells containing antigens. Results areshown as the mean value of spots obtained from triplicate wells.

Analysis of Serum Antibody Titers in Macaques.

Levels of IgG, IgG1, IgG2, IgG3, IgA and IgE antibodies against rBmHSP,rBmALT2, rBmTSP or rBmHAT were determined in the sera (collected onemonth after the final dose of vaccine) of each Rhesus macaque using anindirect ELISA as described herein. Briefly, wells of a 96-wellmicrotiter ELISA plates were coated with 100 ng/well of antigens(rBmHSP, rBmALT2, rBmTSP or rBmHAT) in 0.05 M carbonate-bicarbonatebuffer, pH 9.6. The wells were blocked with 3% BSA in 0.05% PBS-TWEEN 20(PBS-T), and 100 μl of sera samples (diluted in the range of1:100-1:50,000 in PBS-T) from each macaque were added to each well. Goatanti-monkey IgG antibodies conjugated to peroxidase (RocklandImmunochemicals, Gilbertsville, Pa.) was used as secondary antibodies todetermine IgG titer antibodies. The color was developed using OPDsubstrate and absorbance was read at 492 nm in the ELISA reader (BioRad,Hercules, Calif.). To determine the levels of isotype antibodies,biotinylated anti-monkey IgG1 (1:2000), IgG2 (1:200), IgG3 (1:2000), IgA(1:2000) and IgE (1:1000) antibodies (NHP Reagent Resources, Boston,Mass.) were used as secondary antibodies. After washing the plates,optimally diluted streptavidin conjugated horse radish peroxidase (HRP)was added and further incubated for 60 minutes at room temperature andthe color was developed.

ADCC Assay.

PBMC were prepared from heparinized whole blood from a naive healthyanimal as described above. Briefly, ten B. malayi L3 (suspended in 50 μlRPMI 1640 medium containing 10% FBS) were incubated with 2×10⁵ PBMC (in50 μl RPMI 1640) and 50 μl of serum from each animal (collected onemonth after the final dose of vaccine) in a 96-well round bottom tissueculture plate. Five replicates were performed for each serum sample.Control wells contained B. malayi L3 incubated in media, with sera aloneor cells alone. The plates were incubated at 37° C. with 5% CO₂ for 48hours. Following incubation, B. malayi L3 were examined under amicroscope at 24 and 48 hours to determine larval viability. Dead L3were defined as those having a limpid or straight appearance with nomovements for an additional observation period of 8 hours at 37° C. Livelarvae were active, coiled and motile. The percentage larval death wasexpressed as the ratio of the number of dead L3 to that of the totalnumber recovered within the experimental period multiplied by 100.Average larval death in 5 wells were calculated and expressed as percentprotection in each animal.

Knott Test to Determine Microfilaremia (Mf) in Macaques.

The presence of Mf in the blood of macaques was detected using the Knotttechnique as described previously (Liu, et al. (1989) J. Trop. Med. Hyg.92:93-96). Peripheral blood of macaques was screened weekly for Mfstarting from 5 weeks to 20 weeks post challenge. Briefly, whole bloodwas mixed with 9 ml of a 2% formalin solution (prepared in PBS) in a 15ml conical centrifuge tube. The tubes were gently rocked for 2 minutesat room temperature and centrifuged at 1,500 rpm for 5 minutes. Thesupernatant was then thoroughly decanted by turning the tube completelyupside down to remove all the liquid. Following this 5 ml of ACK lysisbuffer (Quality Biologicals, Gaithersburg, Md.) was added to the pelletand the tube was vortexed. Two to three drops of methylene blue solution(Fisher Scientific, Hannover Park, Ill.) was then added to the tubes,gently mixed, and smeared onto five glass slides. The samples wereallowed to dry and read under a microscope using 40× lens objective. Acomparison of Mf counts in blood collected from the saphenous andfemoral veins showed similar results.

Detection of Mf in the Peripheral Blood by PCR. PCR-based assays aremore sensitive in detecting the presence of Mf in the blood samples(Mishra, et al. (2005) Acta Trop. 93:233-7; Tao, et al. (2006) J. Clin.Microbiol. 44:3887-93). Therefore, the PCR based assay was also used toconfirm the presence of Mf in the blood samples of all macaques 20 weeksafter challenge. Whole blood samples were centrifuged at 10,000 rpm for5 minutes and the supernatant containing serum was stored at −20° C. DNAwas isolated from the pellet using DNEASY Blood & Tissue Kit (Qiagen,Valencia, Calif.) according to the manufacturer's instruction. Primerswere synthesized at Integrated DNA Technologies Inc., (Coralville, Iowa)for HhaI tandem repeats. Primer sequences for HhaI tandem repeats were:Forward 5′-GCG CAT AAA TTC ATC AGC-3′ (SEQ ID NO:75) and Reverse 5′-GCGCAA AAC TTA ATT ACA AAA GC-3′ (SEQ ID NO:76). PCR parameters wereinitial denaturation of 94° C. for 5 minutes, followed by 40 cycles of 1minute at 94° C., 1 minute at 56° C., 1 minute at 72° C. and a finalextension of 10 minutes at 72° C. Following PCR reaction, 10 μl of eachPCR product was analyzed on a 1% agarose gel.

PBMC Proliferations Assay.

PBMC collected 10 weeks post-challenge were cultured in 96-well tissueculture plates at a concentration of 1×10⁶ cells/well in RPMI 1640supplemented with 10% FCS. Cells were stimulated either with rBmHATantigen (1 mg/ml) or Concanavalin A (1 mg/ml) or with medium alone(unstimulated) in triplicate wells. PBMC were stimulated in triplicatewells and the plates were incubated at 37° C. in 5% CO₂. After 72 hours,cell proliferation was measured using cell counting kit (CCK-8) (DojindoMolecular Technologies, Inc., Gaithersburg, Md.). Stimulation index ofPBMC proliferation was calculated using the formula: Absorbance ofstimulated cells/Absorbance of unstimulated cells.

Statistical Analysis.

Data are represented as the mean±standard error. One-way ANOVA tests(Kruskal-Wallis) was performed for the antibody titer and T cellproliferation using GraphPad Prism software. Student T test wasperformed for protection studies. A probability (P) value of 0.001 wasconsidered statistically significant.

rBmHAT Vaccination does not Induce any Adverse Reactions in Macaques.

The injection sites were monitored closely for signs of any adversereactions (redness, swelling, etc.) for 7 days post-immunization. Therewere no adverse reactions in any of the vaccinated or control animals.Clinical monitoring showed no dramatic loss of body weight (>10% of theoriginal weight), changes in eating habits or any other behavioralchanges. Temperature measurements obtained daily following immunizationsdid not show any significant variations. Temperature measurements werealso performed at regular intervals using implanted transponders. Therewere no significant variations in the body temperature in vaccinated andcontrol animals.

The lymph nodes in the left and right leg of all animals were monitoredweekly starting approximately 2 weeks prior to challenge (to establish abaseline) and throughout the challenge period. The lymph nodes weremeasured with a caliper and observed for edema. The measurements showedan overall increase in the mean size of the inguinal lymph nodes in bothlegs during the 5-8 week post-challenge period in all groups. Comparedto the baseline (14.5 mm) the lymph node size in control animals were22±1 mm and rBmHAT group were 26.2±1 mm. Following this period, thesizes of the lymph nodes decreased to near pre-challenge levels in allmacaques.

Challenge with B. malayi L3 did not alter the body temperature inmacaques. Analyses of the serum chemistry and hematology (CBC) valuesshowed that they were all in the normal range for all cell types exceptfor a slight increase in the eosinophil counts following L3 challenge ininfected animals.

All Three Antigens in the Multivalent Immunogenic Construct wereImmunogenic in Macaques.

Analysis of the IgG antibody titer in vaccinated macaques showed thatall the macaques developed high titers (1:40,000) of IgG antibodiesafter third immunization against rBmHAT. The titer of antibodies againsteach of the three component antigens in the vaccine construct was thenanalyzed. All macaques developed high titers of IgG antibodies againstrBmHSP12.6 (1:16,000), rBmALT2 (1:24,000) and rBmTSP-LEL (1:16,000).There were slight individual variations in the titer of antibodiesbetween each vaccinated macaque. On a comparative basis, macaque #5242,#5258 and #5259 showed the highest titer of IgG antibodies against thecomponent antigens (except anti-rBmHSP12.6 antibodies in macaque #5258and anti-rBmTSP antibodies in macaque #5259). Macaque #4996 and 5254developed only low titers of antibodies to rBmALT2 and rBmTSP (Table14).

TABLE 14 Antibody Titer Animal ID rBmHSP12.6 rBmALT2 rBmTSP rBmHAT 49966400 3200  16000* 40000 5242 16000* 24000** 16000* 40000 5254 6400 80012800* 40000 5258 6400 24000** 16000* 40000 5259 16000* 24000** 640040000 Macaques were immunized with 200 μg of rBmHAT with alum adjuvant.Anti-rBmHAT antibodies against rBmHSP12.6, rBmALT2, rBmTSP LEL or rBmHATwere evaluated. Each animal differed in the antibody titer against eachantigen. *P < 0.05 and **P < 0.001 statistically significant antibodyIgG antibody titer compared to other animals.

Isotype analysis showed that nearly all of the antibodies were of IgG1isotype against all the four antigens tested (rBmHSP, rBmALT2, rBmTSPand rBmHAT). Levels of IgG2, IgG3, IgA and IgE did not show anysignificant difference from the background values.

rBmHAT Responding Cells were Present in the PBMC of Immunized RhesusMacaques.

To determine the antigen specific proliferative responses, PBMC wascollected four weeks after the final vaccination. Cell proliferation wasdetermined after stimulating CFSE labeled PBMC with rBmHAT proteins for5 days and counting the labeled cells in a flow cytometer. These resultsshowed that the proliferation frequency of antigen-responding cells inthe immunized animals were 3-fold higher (stimulation index 6.1±0.86)compared to the control animals (stimulation index 2.2±1.42). Asexpected, PBMC from all the animals showed robust proliferativeresponses (stimulation index 87.4±0) upon stimulation with pan-Tmitogen, PHA. PBMC cultured in control medium had only low-levelproliferation following 5-day incubation. The proliferation frequencyvalue for each sample was obtained by subtracting the medium alonecontrol value.

Frequency of CFSE-labeled CD3+, CD4+ and CD8+ PBMC proliferating inresponse to antigen stimulation were determined by flow cytometry. Thesestudies showed that there was an increase in the proliferation ofantigen-responding T cells in all immunized macaques compared to controlmacaques. Subset analysis showed that in immunized animals approximately12.7% of the antigen responding T cells were CD4+ cells and 7.9% of Tcells were CD8+ subsets. Background proliferation in the presence ofrBmHAT antigen in the PBMC of control animals were 1.4% for CD4+ cellsand 2.3% for CD8+ cells.

Antigen Responding Cells in the PBMC of Immunized Monkeys Secrete IFN-γ.

Antigen responding cells in the spleen of rBmHAT immunized mice andgerbils predominantly secreted high levels of IFN-γ. Therefore, it wasdetermined whether macaques also show a similar response afterimmunization but before challenge. These studies showed that PBMC fromthree immunized macaques (#5242, #5258 and #5259) all secretedsignificant amounts of IFN-γ when stimulated with rBmHAT antigen (Table15). Culture supernatants of PBMC from macaque #4996 and #5254 only hadbackground levels of IFN-γ similar to that of the PBMC from controlmacaques.

TABLE 15 IFN-γ Secretion (pg/ml) Control Animal ID (alum only) Animal IDrBmHAT + alum 4995 0 4996 0 5240 0 5242 62.5 5249 0 5254 0 5252 0 525862.5 5253 0 5259 62.5

Anti-rBmHAT Antibodies in the Sera of Immunized Macaques can Participatein the Killing of B. malayi L3.

To determine the protective ability of anti-rBmHAT antibodies in thesera of immunized macaques, an in vitro ADCC assay was performed.Results showed that the PBMC from vaccinated macaque were able toparticipate in the killing of 35% of B. malayi L3 (Table 16). When serafrom individual macaques were evaluated maximum killing potential in theADCC was 45% in the sera of macaque #5258. Sera from macaque #5242 and#5259 also showed significant killing potential with 38% and 35% killingrespectively. Sera from macaque #4996 and #5259 had the least ADCCproperty with 25% and 31% killing respectively. No larval death occurredwhen sera from control macaques were used in these assays.

TABLE 16 % Larval Mean % Animal ID Live L3^(a) Dead L3^(a) Death^(a)Larval Death 4995 10 0 0 0 (control) 5240 10 0 0 5249 10 0 0 5252 10 0 05253 10 0 0 4996 7.5 ± 0.6 1.5 ± 0.6  25 ± 5.2* 35% ± 6.1* (immunized)5242 6.5 ± 0.6 4 ± 0.6 38 ± 6.9* 5254 6.5 ± 1  3 ± 0.6 31 ± 7.4* 52586.5 ± 1.5 5 ± 0.6 45 ± 6.3* 5259  7 ± 1.2 3.5 ± 1.2   35 ± 11.5*^(a)Results are presented as Mean ± SD of five wells. Significant larvaldeath *(P < 0.05) compared to other macaques. Control wells were L3incubated with media, cells alone or sera alone.

Immunization with rBmHAT Conferred Partial Protection in Macaques.

One month after the final vaccination, all 10 monkeys were challengedwith 500 B. malayi L3 and screened for the appearance of Mf in theperipheral blood circulation. A Knott test and PCR analysis were used todetect Mf. The Knott test was performed weekly from week 5post-challenge until the animals became positive. In these studies,challenged macaques became positive for Mf starting from week 10post-challenge. During weeks 11-20 post challenge, three of the controlmacaques became positive for Mf. Unfortunately, the remaining twocontrol macaques remained negative through the end of the study. In thevaccinated group, three of the macaques (#5242, #5254 and #5259)remained negative throughout the study. However, two of the vaccinatedmacaques (#4996 and #5258) became positive for Mf. To further confirmthe infection, a PCR analysis was performed, where Hha1 antigen-specificprimers were used to amplify for the presence of Mf-specific DNA in theblood of infected monkeys. PCR analysis confirmed infections in macaque#5249 and #4996. The other three positive animals identified by Knotttechnique were negative by PCR.

rBmHAT Responding Cells were Present in the PBMC of Immunized RhesusMacaques after Challenge.

PBMC collected 10 weeks post challenge was stimulated with rBmHAT todetermine the antigen-specific T cell response. PBMC of three animals#5242 (S.I.—0.928±0.01), #5258 (S.I.—1.091±0.16) and #5256(S.I.—1.0181±0.13) from the vaccinated group that were negative for Mfshowed significant proliferation upon rBmHAT stimulation. Whereas, twoof the vaccinated animals #4996 (S.I.—0.258±0.12) and #5254(S.I.—0.379±0.03) positive for Mf did not show significant proliferationupon rBmHAT stimulation. No significant proliferation was observed inany of the control animals #4995 (S.I.—0.280±0.03), 5240(S.I.—0.415±0.09), 5249 (S.I.—0.300±0.26), 5252 (S.I.—0.507±0.03) or5253 (S.I.—0.475±0.25). S.I of PBMC stimulated with Concanavalin was inthe range of 2.0-3.8.

Eosinophil Numbers were High in Infected Macaques Showing Mf.

Microfilaremic individuals show high eosinophil counts in their blood(Pearlman, et al. (1993) Exp. Parasitol. 76:200-8; Pearlman, et al.(1993) J. Immunol. 151:4857-64). A similar finding was observed inrhesus macaques as well. Absolute counts of eosinophils were determinedon weeks 13, 9, and 5 prior to challenge, on the day of challenge and onweeks 1, 5, 10, and 14 post-challenge. The results showed that there wasan increase in the frequency of eosinophil numbers in the peripheralblood of microfilaremic macaques around 10 weeks post-challenges. Onemacaque (#5259) that was negative for Mf also showed some eosinophilia.Eosinophil counts were 10-fold higher in control macaques that hadmicrofilariae in their peripheral blood.

High Titer of Antigen-Specific IgG Antibodies and ElevatedAntigen-Specific Secretion of IFN-γ from PBMC Correlated with Protectionin the Immunized Macaques.

Since two of the macaques in the immunized group showed presence ofinfection following challenge, vaccine-induced immune responses werecompared in the two infected macaques with similar responses in thethree uninfected macaques within the immunized group. Values before andafter challenge were compared. Values before challenge eliminated anybias due to the challenge of parasites. Comparative immunological valuesare presented in Table 17.

TABLE 17 PBMC Proliferation, Mean S.I. ± S.D. (n = 3) StimulatedStimulated Macaque Group Animal ID with ConA with rBmHAT Control 49953.260 ± 0.01 0.280 ± 0.03 (immunized 5240 3.090 ± 0.58 0.415 ± 0.09 withalum) 5249 2.982 ± 0.24 0.300 ± 0.26 5252 3.674 ± 0.83 0.507 ± 0.03 52532.582 ± 0.72 0.475 ± 0.25 rBmHAT 4996 3.874 ± 0.47 0.258 ± 0.12(immunized 5242 2.170 ± 0.43   0.928 ± 0.001** with rBmHAT + 5254 2.068± 0.18 0.379 ± 0.03 alum) 5258 3.304 ± 0.64  1.091 ± 0.16** 5259 2.883 ±0.27  1.0181 ± 0.13** **Significant proliferation of PBMC **(P < 0.001)compared to PBMC from other macaques.

Results showed that the titer of IgG antibodies was significantly highin the three immunized macaques that did not develop the infection afterthe challenge. Similarly, PBMC from the same three macaques secretedhigher levels of IFN-γ when stimulated with the rBmHAT antigen. PBMCfrom the two immunized macaques that developed the infection afterchallenge were unable to secrete similar levels of IFN-γ in response torBmHAT stimulation. An ELISPOT assay was performed using PBMC fromvaccinated and control macaques. Results showed that in all the infectedmacaques there was a significant increase in the number ofantigen-specific IL-10 secreting cells compared to IFN-γ secretingcells. When the ratios of IFN-γ to IL-10 secreting cells in the PBMC ofimmunized macaques were compared, there was a significant increase inthe IL-10 secreting cells in the two vaccinated macaques that showedinfection (Table 18). These findings suggest a clear correlation betweenthe type immune responses elicited and the failure to establishinfection in the vaccinated macaques.

TABLE 18 Immunological Immunological values before L3 values after L3challenge challenge Antibody titer Ratio of of >12,000 IFN-γ:IL-10Animal rBmTSP secreting ID rBmHSP rBmALT2 LEL IFN-γ Mf cells 4995^(a) −− − − + 1:3 5240^(a) − − − − − 1:1 5249^(a) − − − − +  1:11 5252^(a) − −− − −   1:0.01 5253^(a) − − − − +  1:13 4996^(b) − − + − + 1:45242^(b) + + + + −    1:0.003 5254^(b) − − + − + 1:2 5258^(b) + + + + −   1:0.001 5259^(b) + + − + −   1:0.02 ^(a)Control, immunized with alum.^(b)rBmHAT, immunized with rBmHAT + alum.

Example 8: Valency Comparisons

Monovalent, bivalent and trivalent vaccination trials of recombinantheat shock protein 12.6 (rHSP12.6), abundant larval transcript-2(rALT-2) and tetraspanin large extracellular loop (rTSP-LEL) proteinswere compared. Recombinant proteins were prepared as described herein.The bivalent immunogenic compositions and multivalent immunogeniccompositions (SEQ ID NO:70) were produced as fusion proteins. Mice (N=5)were immunized subcutaneously using a protein prime-boost vaccineregimen. Immunized and control animals were challenged with live thirdstage infective larvae (L3) of B. malayi using a micropore chambermethod. After 48 hours of implantation, animals were sacrificed and thechambers were recovered from peritoneal cavity. Contents of each chamberwere emptied and larvae were examined microscopically at 100× to assesslarval death. The results of this analysis are presented in Table 19.

TABLE 19 Percent Larval Death Group Protein Vaccine (protection) ControlAlum  9 ± 3.4 Monovalent rHSP12.6 (rH) 58 ± 7.8 rALT-2 (rA) 78 ± 3.7rTSP LEL (rT) 49 ± 2.2 Bivalent rHA 81 ± 6.5 rAT 72 ± 1.1 rHT 68 ± 4.4Multivalent rHAT 95 ± 3.1

The results indicate that the multivalent immunogenic compositionsynergistically enhanced the protective immune responses in vaccinatedanimals compared to monovalent and bivalent compositions.

B. malayi parasite does not mature into adults in mice. However,vaccine-induced protection against adult worm establishment can bedetermined in jirds. Therefore, monovalent, bivalent and trivalentvaccines were evaluated in jirds. Animals (N=10) were immunizedsubcutaneously with recombinant proteins. Jirds were challenged with 100B. malayi L3s and worm establishment was determined on day 95 afterchallenge. Percent protection values were calculated as the percentreduction in worm establishment compared with control jirds. The resultsof this analysis are presented in Table 20.

TABLE 20 Group Protein Vaccine Percent Protection Control Alum 15.2 ±3.3 Monovalent rHSP12.6 (H)  70.0 ± 12.6 rALT-2 (A) 72.7 ± 8.8 rTSP LEL(T) 68.1 ± 2.4 Bivalent rHA 83.3 ± 3.3 rAT  77.1 ± 12.3 rHT  70.2 ± 11.8Multivalent rHAT 90.2 ± 9.1

The results indicate that the multivalent immunogenic compositionsynergistically enhanced the protective immune responses in vaccinatedanimals compared to monovalent and bivalent vaccines.

Example 9: Vaccine Comparisons

Monovalent, bivalent and multivalent immunogenic compositions of thisdisclosure were compared in mice, jirds and mastomys. Animals wereimmunized as described, challenged with B. malayi L3 and wormestablishment was determined. The results of these analyses arepresented in Table 21. Of note, rBmHAX immunization gave 98% protectionin mice and 97% protection in jirds. These findings show

TABLE 21 Mice* Jirds Mastomys Group Test Control Test Control TestControl rWbALT2^(a) 73 + 3.7% 2 + 0%  73 + 1%  0 + 1% 71.66 + 8.8% 4.2 +1.3% rBmHSP^(a) 58 + 7.8% 0 + 0%  61 + 0%  4 + 0%  69.97 + 12.6% 2.1 +0.2% rWbTSP^(a) 49 + 2.2% 3 + 1%  33 + 2%  1 + 1% 68.13 + 2.4% 1.1 +1.1% rBmTPX^(a) 48 + 2.1% 0 + 0%  52 + 2.5% 0 + 0% ND ND rWbGST^(a) 49 +3.1% 2 + 1%  61 + 1%  0 + 0% ND ND rWbHA^(b) 81 + 6.5% 3 + 3.2% ND ND83.25 + 3.3% 7.2 + 1.1% rWbAT^(b) 72 + 1.1% 1 + 2.1% ND ND  77.13 +12.3% 5.4 + 2.3% rWbHT^(b) 68 + 4.4% 6 + 3.8% ND ND  70.23 + 11.8% 7.1 +3.3% rBmAX^(b) 74 + 3.3% 0 + 0%  80 + 3.5  0 + 0% ND ND rWbGA^(b) 68 +2.5% 2 + 4.1% 72 + 3.3% 0 + 0% ND ND rBmHAT^(c) 98 + 2.1% 4 + 3.3% 95 +3.5% 2 + 1% 95.23 + 9.1% 4.4 + 1.2% rBmHAX^(c)** 98 + 1.2% 3 + 1.0% 97 +2.1% 0 + 0% ND ND ^(a)Monovalent vaccine. Wb, W. bancrofit. Bm, B.malayi. ^(b)Bivalent vaccine. H, HSP. A, ALT2. T, TSP. X, TPX. G, GST.^(c)Trivalent vaccine. *Animals were immunized s/c with four injectionsof 15 μg of the vaccine antigen plus 15 μg of alum at 2-week intervals.Test animals were challenged with 100 L3 and worm establishment wasdetermined on day 90 post-challenge. The micropore chamber challengemethod was used in mice. In this method, 20 L3 were placed in amicropore chamber, which was implanted into the peritoneal cavity. After48 hours the chambers were removed to determine live and dead larvae.Data mean + SD. N=10. **Mice and jirds were immunized with 15 μg ofrBmHAX plus 15 μg of alum with a total of four immunizations at 2 weeksinterval. Blood was collected on day 0, 14, 28, 42, 49 and 70 to monitorthe titer of antibodies against each of the component antigens. Thefollowing titers were observed on day 49 (ALT-2 1:60,000; HSP 1:40,000,TPX 1:40,000). All the animals were challenged on day 49 with 20 B.malayi L3 for mice and 100 B. malayi L3 for jirds. Worm establishment orworm death in immunized animals was observed at 48 hours after surgicalimplantation of L3 in mice or 90 days after infection in jirds. Percentprotection was calculated as described herein.

Example 10: Tetravalent Fusion Protein (rBmHAXT) Vaccine Antigen AgainstLymphatic Filariasis in a Mouse Mode

Cloning, Expression and Purification of rBmHAXT Recombinant Protein.

GenScript (Piscataway, N.J.) supplied the sequences of bmhspl2.6(GENBNAK Accession No. AY692227.1), bmalt-2 (GENBNAK Accession No.JF795950.1), bmtpx-2 (GENBNAK Accession No. AF319997.1) and bmtsp(GENBNAK Accession No. JF795955.1) in the pUC57 vector. The genes wereamplified using forward 5′-CGG GAT CCA TGG AAG AAA AGG TAG TG-3′ (SEQ IDNO:31) and reverse 5′-CCC GAA TTC TTA ATG TTT CTC AAA ATA TGC TTT-3′(SEQ ID NO:89) with restriction sites for BamHI and EcoRI. ThePCR-amplified products were cloned into the pRSETA expression vector,transformed into competent BL21 (DE3) Escherichia coli cells forexpression of the recombinant proteins with 6× histidine tag.Recombinant fusion proteins were purified using immobilized metalaffinity Ni⁺-charged agarose chromatography column sold under thetradename SEPHAROSE® (GE Healthcare Life Sciences, Pittsburgh, Pa.) andeluted with 300 mM imidazole. Endotoxin in the final purified proteinpreparation was removed using an endotoxin removal column (Thermo FisherScientific, Rockford, Ill.). The expression and purity of recombinantproteins was confirmed in 12% SDS-PAGE gel and western blot usinganti-His antibodies (Qiagen, Valencia, Calif.). Protein concentrationwas determined using a Bradford reagent (Thermo Fisher Scientific).

Adjuvants.

Three different adjuvant formulations were used with recombinant BmHAXT.Alum (AL007) and Alum plus GLA, a synthetic TLR4 agonist (AL019) waspurchased from the Infectious Disease Research Institute, Seattle, Wash.and Mannosylated Chitosan (MCA) was a gift from Pacific GeneTech, HongKong.

Animals and Parasite.

Six to eight weeks old Balb/c mice purchased from Taconic biosciences(Hudson, N.Y.) were used in these experiments. Use of animal in thisstudy was approved by the animal care committee of the University ofIllinois, Rockford following the National Institutes of Healthguidelines for the care and use of laboratory animals. The infectivelarval stage (L3) of B. malayi was obtained from the NIAID/NIHFilariasis Research Reagent Resource Center (University of Georgia,Athens, Ga.).

Immunization of Balb/c Mice.

For the immunization, mice were randomly divided into seven groups offive mice per group: (1) rBmHAXT+AL007 given s/c, (2) rBmHAXT+AL019given s/c, (3) rBmHAXT+MCA (first dose s/c and booster doses givenorally), (4) AL007 control given s/c, (5) AL019 control given s/c, (6)MCA control (first dose s/c and booster doses given orally), (7)rBmHAXT+MCA control (all doses were given orally). Each mouse receivedthree doses of 15 μg of rBmHAXT and 15 μg of respective adjuvantformulation at 15 days interval.

Collection of Serum, Peritoneal Fluid and Spleen.

Blood samples were collected from the submandibular vein of each mouseon day 0 (pre-immune), and then two weeks after each immunization andkept at room temperature for 1 hour to clot. Serum was separated, andaliquots were kept frozen at −80° C. for further use. Peritoneal cavitywas washed with 500 μl of sterile saline solution and the fluid wascollected from each mouse and processed. Spleen was then collected fromeach animal, washed three times with complete RPMI-1640 mediumsupplemented with 10% FBS and 1× antibiotic/mycotic solution (Sigma, St.Louis, Mo.).

Titer of IgG Antibodies in the Serum and Peritoneal Fluids.

The titers of rBmHAXT-specific IgG antibodies in the sera samples and inthe peritoneal fluids were evaluated using an indirect ELISA. Wells werecoated with 1 μg/ml of rBmHAXT overnight at 4° C. After washing andblocking of the plates, diluted (1:100, 1:1,000, 1:5,000, 1:10,000,1:20,000 and 1:40,000) sera or peritoneal fluid samples were added andincubated for 1 hour at room temperature. HRP-conjugated chickenanti-mouse IgG antibodies (Thermo Fisher scientific) were used as thesecondary antibodies and color was developed using the 1-step UltraTMB-ELISA substrate (Thermo Fisher Scientific) The reaction was stoppedusing 0.16 M H₂SO₄, and optical density was determined at 450 nm in aBioTek Synergy 2 ELISA reader.

Levels of Antigen Specific Antibody Isotypes in the Serum and PeritonealFluids.

Levels of rBmHAXT-specific antibody isotypes (IgG1, IgG2a, IgG2b, IgG3,IgE, IgM) were determined in the sera and peritoneal fluid samples usingan indirect ELISA. Respective isotype-specific biotinylated goatanti-mouse antibodies (Sigma) and streptavidin-HRP (1:20,000) were usedas the secondary antibodies. Color was developed with 1-step Ultra-TMB.The reaction was stopped using 0.16 M H₂SO₄, and optical density wasdetermined at 450 nm in a BioTek Synergy 2 ELISA reader.

Challenge Studies.

To determine vaccine-induced protection, a micropore chamber challengemethod was used as described previously (Dakshinamoorthy, et al. (2013)Vaccine 31(12):1616-22). Briefly, 20 L3s of B. malayi were placed in amicropore chamber and surgically implanted into the peritoneum of eachmouse. Seventy-two hours after implantation, the micropore chambers wererecovered. Contents of each chamber were emptied and larvae were countedand examined microscopically for adherence of cells and for larvaldeath. Larvae that were clear, straight and with no movement werecounted as dead. Larvae that were active, coiled and translucent werecounted as live. The percentage of protection was expressed as thenumber of dead parasites/number of total parasites recovered×100.

Levels of Secreted Cytokines in Culture Supernatant of Splenocytes.

A single cell suspension of spleen cells was prepared and stimulatedwith 1 μg/ml of rBmHAXT or ConA. Unstimulated spleen cells were kept asnegative control for the assay. After a 72-hour incubation, culturesupernatants were collected and levels of IL-2, IL-4, IL-6, IFNγ, TNFα,IL-10, and IL-17A were determined using a cytokine bead array kit (BDBio Sciences, San Jose, Calif.).

Analysis of T Cell Subsets by Flow Cytometer.

Spleen cells from the above cultures were then washed and labeled withfluorescent labeled anti-mouse CD3 (APC), CD4 (PE) and CD8 (PE/cya7) andthe percent population of each cell type was determined in a flowcytometer. Briefly, cells were incubated with FcγII receptor blocker instaining buffer (2% FBS+0.1% sodium azide) for 30 minutes at 4° C. withsubsequent wash in staining buffer. All three fluorescent-labeledantibodies were added to the cells and incubated for 1 hour at 4° C. inthe dark. After washing with staining buffer, cells were fixed in 4%paraformaldehyde and analyzed in a BD FACSCalibur™ (BD Biosciences) flowcytometer.

Another set of cells from the above experiment was stained with CD3(APC) and within the CD3-gated population, the CD62L (PE/Cya7) and CCR7(PE) positive T cells were identified as T-central memory cells. Thecell population was also stained for intracellular IFN-γ (FITC) todetermine the percent of IFN-γ positive T-central memory cells.

Statistical Analysis.

Data presented are mean±standard deviation (SD). Statisticalsignificance of mean differences among different sample groups wasanalyzed using non-parametric Kruskal-Wallis test followed by Bonferronicorrection for multiple tests using SPSS software (v24.0, IBM, NY). Thesignificance level was defined as P<0.05.

Titer of rBmHAXT-Specific IgG Antibody.

Recombinant BmHAXT protein was prepared and expressed. On the SDS-PAGEgel, the molecular mass of rBmHAXT was approximately 60 kDa and appearedas a single band. Endotoxin levels in the final purified preparationswas <3 EU/0.1 mg of protein. The titer of rBmHAXT-specific IgG antibodywas high (1:20000) in the sera of the rBmHAXT+AL007 group and in therBmHAXT+AL019 group (p<0.05). However, the titer was less (1:10000) inrBmHAXT+MCA group. In rBmHAXT+MCA group where all the doses were givenorally, there was very little titer of antigen-specific antibodies,nearly same as AL007, AL019 and MCA adjuvant control groups. Similarly,when the peritoneal fluids were analyzed, high titer of antigen-specificIgG antibody in rBmHAXT+AL019 and rBmHAXT+AL007 vaccinated animals wereobserved compared to rBmHAXT+MCA group. The titer of IgG antibodies wasless in the peritoneal fluids when compared to the respective serasamples from the same animals.

Antibody Isotypes in Serum and Peritoneal Fluid.

To determine the type of humoral immune response generated againstrBmHAXT, the antibody isotypes IgG1, IgG2a, IgG2b, IgG3, IgE, IgM andIgA were determined in serum and peritoneal fluid samples. The resultsshowed that IgG1 was the predominant isotype of antibodies in allvaccinated groups (p=0.0001) except rBmHAXT+MCA (all oral dose) group,which was similar to the controls. Titer of IgG2a and IgG2b antibodieswere also significantly high in all vaccinated animals (p<0.05) comparedto controls except in rBmHAXT+oral MCA vaccinated group. Titer of IgG3,IgE, IgM, and IgA antibodies in the vaccinated animals did not show anysignificant changes compared to the controls. Significantly, high titersof IgG1 antibodies were present in the peritoneal fluids ofrBmHAXT+AL007 and rBmHAXT+AL019 vaccinated animals (p=0.0001) comparedto rBmHAXT+MCA subcutaneous group and controls. Titer of IgG2a and IgG2bantibodies in the peritoneal fluid of all vaccinated animals were notsignificantly different from the controls.

Vaccination with rBmHAXT+AL019 Conferred Maximum Protection.

Vaccine-induced protection was determined using a micropore chamberchallenge method. The results showed that maximum protection wasobserved in animals vaccinated with rBmHAXT+AL019 (88.05±3.9%; p=0.0001)followed by rBmHAXT+AL007 (79.47±2.6%; p=0.0001) and rBmHAXT+MCA(78.67±5.47%; p=0.0001). Several cells were found attached to thesurface of the dead larvae. These results indicate that AL019 may be abetter adjuvant for rBmHAXT compared to AL007 (p=0.0037) and MCA(p=0.02). Vaccination with rBmHAXT+oral MCA conferred only 17.97±5.75%,which was similar to the adjuvant control groups (AL007, 20.55%; AL019,24.19%; and MCA, 15.742%).

Spleen Cells from rBmHAXT-Vaccinated Animals Secreted Both Th1 and Th2Cytokines.

Cytokines level in the culture supernatants of spleen cells weredetermined using a cytokine bead array. The results showed that secretedlevels of Th1 (IFN-γ, IL-2, IL-6, IL-17A) and Th2 (IL-4 and IL-10)cytokines were significantly (p<0.05) increased in the culturesupernatants of spleen cells from rBmHAXT+AL019 and rBmHAXT+AL007vaccinated animals compared to the respective adjuvant control animals.Spleen culture supernatants from rBmHAXT+MCA vaccinated animals hadsignificantly high levels of IFN-γ (p=0.01) and IL-6 (p=0.0001) comparedto the respective adjuvant control. However, there was no significantdifference in the levels of the other cytokines measured. Cytokinelevels in the culture supernatants from rBmHAXT+oral MCA group weresimilar to the adjuvant controls.

T_(CM) Cells were Generated in the Spleen of rBmHAXT-Vaccinated Animals.

Spleen cells were cultured at 37° C. for 72 hours, stimulated with 1μg/ml of rBmHAXT protein. After 72 hours, cells were harvested andstained with CD3/CD4/CD8 antibodies and evaluated via flow cytometer.There was a slight but significant increase in the CD8⁺ cell populationin the rBmHAXT+AL019-treated group (p≤0.05) compared to the othergroups. To determine the percent of T_(CM) cells in the spleen,splenocytes were stained with CD62L/CCR7 antibodies and analyzed in aflow cytometer. Cells that were dual positive for CD62L/CCR7 wereconsidered to be T_(CM) cells. The results showed that rBmHAXT-treatedanimals showed high percentage of T_(CM) cells irrespective of theadjuvant used (p≤0.001).

T_(CM) Cells were Predominantly IFNγ⁺. IFN-γ secreting T_(CM) cells arebelieved to play a major role in vaccine-induced protection in parasiticinfections (Maggioli, et al. (2016) Front. Immunol. 7:421). Therefore,the percentage of CD62L⁺ CCR7⁺ T_(CM) cells that expressed intracellularIFN-γ was measured. The results showed that cells fromrBmHAXT+AL019-vaccinated animals had a significantly (p<0.01) highpercentage of IFNγ⁺ T_(CM) cells compared to rBmHAXT+AL007- andrBmHAXT+MCA-vaccinated groups.

Example 11: Prophylactic Vaccine Against Human Lymphatic Filariasis inNon-Human Primates

Non-Human Primates. Forty male or female disease-free rhesus macaques (3to 5 years old) were purchased from PrimGen (Hines, Ill.) and housed atthe Bioqual's facility at Rockville, Md. All animals were screened forthe absence of filarial infections prior to enrolling them in the studyby analyzing the blood for the presence of microfilarial Hha-1 by PCR(Hoti, et al. (2003) Acta Trop. 88:77-81; Rao, et al. (2006) J. Clin.Microbiol. 44:3887-3893) and serum for the presence of antibodiesagainst rBmSXP-1 (Vasuki, et al. (2003) Acta Trop. 86:109-114; AbdulRahman, et al. (2007) Filaria J. 6:10) and rBmHAXT proteins wereanalyzed using enzyme-linked immunosorbent assay (ELISA). Animals thatwere positive for any of the proteins were not enrolled in the study.

Parasites.

Brugia malayi infective third stage larvae (L3) were obtained from theNIAID/NIH Filariasis Research Reagent Resource Center (University ofGeorgia, Athens, Ga.).

Adjuvants.

Two different adjuvants were compared in this study. Alum (AL007) andAlum plus a synthetic TLR4 agonist GLA (AL019) purchased from InfectiousDisease Research Institute, Seattle, Wash.

Cloning and Expression of Multivalent Recombinant Proteins.

rBmHAT protein was expressed in Escherichia coli BL21 (DE3), purifiedand analyzed as described herein. The coding sequence (CDS) ofmultivalent fusion protein rBmHAT (composed of bmhsp 12.6, bmalt-2 andbmtsp) and rBmHAXT (composed of bmhsp 12.6, bmalt-2, bmtpx2 and bmtsp)were synthesized at GenScript (Piscataway, N.J.). The sequences wereprovided in pUC51 vector. Both CDS were PCR amplified using the samegene specific primers (Forward primer: 5′-CGG GAT CCA TGG AAG AAA AGGTAG TG-3′, SEQ ID NO:31 & Reverse primer: 5′-CGG AAT TCT CAA TCT TTT TGAGAT GAA T-3′, SEQ DI NO:36) with restriction sites for BamHI and EcoRIand cloned into the expression vector pRSETA (Invitrogen, Carlsbad,Calif.) with the 6× Histidine tag. The ligated constructs for both bmhatand bmhaxt were further transformed into the expression strain of E.coli BL21 (DE3). Expression of recombinant proteins was induced with 1mM IPTG. The recombinant proteins were purified using nickel affinitycolumn chromatography (GE Healthcare Life Sciences, Pittsburgh, Pa.) andthe purity of the recombinant proteins was confirmed in 12% SDS PAGE geland by western blot using anti-penta His antibodies (Qiagen, Velencia,Calif.). Endotoxin in the final prep was removed using an endotoxinremoval column (Thermo Fisher Scientific, Rockford, Ill.). Finalconcentration of rBmHAT and rBmHAXT proteins was determined by Bradfordassay (Qiagen).

Immunization of Macaques.

This was a double-blinded vaccination trial. A total of 40 macaques wererandomly divided into three treatment groups and one control group with10 macaques per group. All the treated animals received four doses of150 μg of the vaccine antigen and 2 mg of the adjuvant on days 0, 28, 56and 84. Treatment group 1 received rBmHAT+alum, treatment group 2received rBmHAT+AL019 and treatment group 3 received rBmHAXT+AL019.Control animals received AL019 adjuvant only. The injection sites weremonitored daily for any adverse reactions (redness, swelling, etc.) forup to 7 days post-immunization. Blood samples were collected prior toeach immunization and before challenge.

Cell Counts, Serum Chemistry and Complete Blood Count (CBC) Analysis.

CBC and serum chemistries were analyzed using commercial automatedhematology and serum chemistry analyzers by IDEXX. Samples collectedprior to the initiation of the study served as a normal referencebaseline for each animal.

Antigen-Specific Antibody Levels in Macaque Sera.

Levels of rBmHAT-, rBmHAXT-, rBmHSP-, rBmALT-2-, rBmTPX-, orrBmTSP-specific total IgG, IgG1, IgG2, IgG3, IgM and IgE antibodies weredetermined in the sera of each Rhesus macaque using an indirect ELISA asdescribed elsewhere herein.

Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Assay.

ADCC assay was performed as described elsewhere herein. Approximatelyten live B. malayi L3 each were incubated at 37° C. with 5% CO₂ intriplicate wells along with 2×10⁵ PBMC and 50 μl of sera samples.Seventy-two hours after incubation, viability of B. malayi L3 wasdetermined. The percentage larval death was expressed as the ratio ofthe number of dead L3 to the total number recovered from each wellmultiplied by 100.

The culture supernatant from the ADCC assay was also collected todetermine the level of myeloperoxidase (MPO) activity using a kitpurchased from Biovision (Milpitas, Calif.) and the values are expressedas mU/minute in per ml of culture supernatant.

Antigen-Specific Proliferation of Peripheral Blood Mononuclear Cells(PBMC).

PBMC (1×10⁷ cells/well in 1 ml) were incubated at 37° C. in the dark for15 minutes with 5 mM of carboxyfluorescein diacetate succinimidyl ester(CFSE) Following incubation, cells were washed, incubated for anadditional 30 minutes at 37° C. and plated at 2×10⁶ cells/well in 1 mlin a 24-well tissue culture plate. Five hundred μl of the medium wasremoved the next day and rBmHAXT (1 μg in 500 μl) was added to thewells. Cells incubated with RPMI medium alone served as negativecontrols and concanavalin A (1 mg/well)-stimulated cells served aspositive controls. Cells were harvested on day five after culture,labeled with anti-CD3-APC antibody, fixed in 4% paraformaldehyde for 10minutes at room temperature and data acquired on a BD FACSCalibur™ flowcytometer and analyzed using ModFit LT software (Verity Software House,Topsham, Me.).

Parallel cultures of cells incubated for 3 days were harvested,paraformaldehyde fixed and labeled with combinations of anti-CD3-APC,anti-CD4-FITC, anti-CD8-PE, anti-CD28-PE, or anti-CCR7-FITC.Intracellular IFN-γ and IL-4 were determined after fixing andpermeabilization. Data was acquired on BD FACSCalibur™ flow cytometerand analyzed using Flow Jo v10.1 (FlowJo, LLC, Ashland, Oreg.).

Secreted levels of cytokines (GM-CSF, IFN-γ, IL-12p70, IL-1β, IL-4,IL-5, IL-6, IL-15, IL-16 and TNF-α) in the cell culture supernatantswere measured using an antibody-based Rhesus Cytokine Quantibody ArrayGS1 (RayBiotech, Inc., Norcross, Ga.) according to manufacturer'sprotocol. The intensity of the fluorescence signals from the slidearrays was scanned and data analyzed after subtracting the backgroundsignals and normalization to positive controls.

B. malayi Infective Larval (L3) Challenge.

One month after the last immunization, all macaques were challengedsubcutaneously with 130-180 B. malayi L3. All the larvae were examinedmicroscopically for their viability, counted, and only the viable larvaewere used for challenge. Following challenge, all the macaques weremonitored routinely for any possible alterations in the clinicalbiochemistry panel (serum chemistry, hematology, complete blood countanalysis and CD4⁺/CD8⁺ T-cell counts), physical parameters (signs ofadverse reactions at the site of challenge, body weights, bodytemperature, body condition, lymphoedema and lymph node measurements)and behavioral patterns.

Confirming the Establishment of Challenge Infection in Macaques.

It is practically difficult to count the number of adult wormsestablished in each macaque. However, several pieces of evidences wereused to confirm the presence of active infection in macaques includingmicroscopic and Hha-I PCR analyses. For microscopic analysis, on week 5,10, 15 and 18 post-challenge, 10 ml of blood was collected from eachmacaque between 6-10 μm and screened for the presence of microfilariaeusing a modified Knott technique. For Hha-I PCR analysis, DNA isolatedfrom 200 μl of blood samples using the Gen Elute blood genomic DNA kit(Sigma-Aldrich) were PCR amplified for Hha-I tandem repeat genes asdescribed previously (Hoti, et al. (2001) Bull. Entomol. Res. 91:87-92)and the amplified PCR products were sequenced to confirm the Hha-Isequence.

Titer of Anti-rBmSXP-1 Antibodies in Sera.

BmSXP-1 is a highly sensitive and specific diagnostic antigen for B.malayi infections. Typically, a single sex infection will not have anyMf in the peripheral circulation. Thus, microscopy and PCR approach maynot detect these dormant infections. However, presence of active worm ininfected subjects or animals can be confirmed by determining the titerof IgG4 antibodies against BmSXP-1 antigen. An ELISA was used todetermine the titer of these antibodies in the sera of macaques. Sinceno reliable commercial anti-macaque IgG4 antibodies were obtainable,titer of anti-rBmSXP-1 IgG antibodies was used as a marker to detectdormant infections.

Lymphoscintigraphy Analysis.

The lymphoscintigraphic analysis was carried out as described elsewhereherein.

Statistical Analysis.

Data presented are mean±standard deviation (SD). Statisticalsignificance of mean±differences among different sample groups wasanalyzed using non-parametric Kruskal-Wallis test followed by Bonferronicorrection for multiple tests using SPSS software (v24.0, IBM, NY). Thesignificance level was defined as P≤0.05. To analyze the vaccine-inducedprotection, Chi-square test was used to compare the proportions acrossthe groups and Fisher's exact test was used where appropriate. Oddsratios (OD) were calculated to determine the differences between groups.

Immunization with the Vaccine Candidates Generated High Titer ofAntigen-Specific IgG Antibodies and Their Isotypes.

Molecular mass of rBmHAT is approximately 39 kDa and rBmHAXT isapproximately 60 kDa. Both proteins were prepared to over 95% puritywith endotoxin levels <10 EU/μg of the protein. Immunization of macaqueswith the purified proteins generated high titer of antigen-specific IgGantibodies. The IgG antibodies were specific to each component of thefusion protein. Maximum IgG antibody titer was achieved after the seconddose of immunization (Table 22) indicating that two doses of vaccine issufficient to achieve maximum antibody titer.

TABLE 22 Macaque Groups Immunizations (n=10) First Second Third FourthrBmHAT + AL007 1:1250 1:20000 1:20000 1:20000 rBmHAT + AL019 1:100001:20000 1:20000 1:20000 rBmHAXT + AL019 1:1250 1:20000 1:20000 1:20000AL019 1:125 1:125 1:125 1:125 rBmHAT, recombinant Brugia malayiHSP12.6 + ALT-2 + TSPLEL; rBmHAXT, recombinant B. malayi HSP12.6 +ALT-2 + TPX-2 + TSPLEL.

IgG1 and IgG2 were the most predominant isotype of IgG antibodies in thesera of all immunized animals. Levels of IgG3 antibodies weresignificantly elevated in the sera of rBmHAXT plus AL019 immunized groupcompared to the control animals. IgM and IgE antibodies were notsignificantly different from controls in the sera of vaccinated animals.

Antigen Responding Memory Cells were Present in the Blood of VaccinatedMacaques.

Proliferation index of PBMCs from vaccinated animals was high inresponse to the respective antigens compared to controls. Cells fromcontrol group replicated only once during the five days in culturecompared to the vaccinated group where the cells divided up to eightgenerations. Evaluation of the memory cell population within thedividing antigen-responding CD4 and CD8⁺ T cells showed that bothCD28⁺CCR7⁻ effector memory T cells (T_(EM)) and CD28⁺ CCR7⁺ centralmemory T cells (T_(CM)) were selectively expanded in rBmHAXT plus AL019and rBmHAT plus AL007 immunized macaques. The T_(EM) cells predominantlywere positive for intracellular IFN-γ and T_(CM) cells predominantlypositive for intracellular IL-4. Analysis of the culture supernatants ofthe PBMCs showed a marked increase in the secreted levels of cytokines(GM-CSF, IFN-γ, IL-12p70, IL-1β, IL-4, IL-5, IL-6, IL-15, IL-16 andTNF-α) compared to AL019 controls. PBMCs from rBmHAXT+AL019 vaccinatedanimals secreted nearly 10-fold higher levels of IFN-γ compared to AL019controls.

Immunization with rBmHAXT Conferred Maximum Protection.

Ten weeks after challenge with B. malayi L3, seven out of 10 controlanimals (70%) showed Mf in their peripheral blood and they continued tobe positive for Mf until completion of the experiment (18 weekspost-challenge). However, only 3 out of 10 macaques (30%) in the rBmHAXTplus AL019 group showed Mf in their blood (FIG. 4). These findings werefurther confirmed by PCR analyses of the blood samples for the presenceof B. malayi-specific Hha-1 and by an ELISA for IgG antibodies againstSXP-1 antigen. Repeat examination of the blood did not show any evidencechallenge of infection in any of the negative animals until completionof the experiment (18 weeks post) indicating that immunization withrBmHAXT plus AL019 conferred 57.14% protection after adjusting the 30%of a microfilaremic macaques in the control group (p=0.073, oddsratio=0.18). Five out of 10 animals in the rBmHAT+AL019 group (p=0.649,odds ratio=0.42) and 7 out of animals in the rBmHAT+AL007 group (p=1.0,odds ratio=1.0) were positive for Mf. Statistical correlation betweendifferent groups showed that rBmHAXT+AL019 conferred better protectionthan other vaccinated groups and AL019 control (rBmHAXT+AL019 vs AL019control—p=0.073; rBmHAXT+AL019 vs rBmHAT+AL019—p=0.649; rBmHAXT+AL019 vsrBmHAT+AL007—p=0.073 and rBmHAT+AL019 vs rBmHAT+AL007—p=0.649). Thus,based on the odds-ratio, the odds that the AL019 control group would bepositive for Mf were 2.33 times higher than the odds that rBmHAT+AL019group would be Mf positive. The odds that the AL019 control group wouldbe positive for Mf were 5.43 times higher than the odds that therBmHAXT+AL019 group would be Mf positive. The odds that the rBmHAT+AL019group would be positive for Mf were 1.95 times higher than the odds thatthe rBmHAXT+AL019 group would be Mf positive. These statistical analysesindicate that protection conferred by rBmHAXT+AL019 immunization wassignificantly better than rBmHAT+AL007 and rBmHAT+AL019 immunizations.

An antibody dependent cellular cytotoxicity (ADCC) assay was alsoperformed as an in vitro surrogate for determining vaccine-inducedprotection. These assays also confirmed that sera samples from macaquevaccinated with rBmHAXT+AL019 were more efficient in killing the B.malayi L3 in vitro compared to the sera samples from the other twovaccinated groups (FIG. 5). Several cells were found attached to thedead larvae. In fact, many of the dead larvae were totally covered bycells within 24 hours after incubation. In this study the morphology ofthe cells was not identified. However, previous studies suggest that thebound cells are predominantly monocytes. To determine the activationstatus of these cells, levels of myeloperoxidases (MPO) in the culturesupernatants from these ADCC assays were measured. The results showed anincrease in the secreted levels of MPO (2.08 to 2.78 mmol/min/ml) in thewells with sera from vaccinated animals and PBMCs compared to the wellswith sera from control animals (1.99 mmol/min/ml), indicating activationof MPO producing cells.

Mf-Negative Vaccinated Animals Did not Develop Lymphatic Pathology.

Adult filarial parasites living within the lymphatic vessels causesevere inflammation leading to lymphedema and blockage of lymph flow.The presence of lymphatic blockage in this study was determined bylymphoscintigraphy. Lymph flow was compared between the right and leftleg in the same animal. Challenge infections were given on the rightleg. The results showed that on week 16 post-challenge, there was asignificant reduction in the lymph flow in the right leg of all Mfpositive animals compared to their left leg indicating lymph blockage.Lymph flow did not show any significant differences between the rightand left leg in all Mf negative animals in the vaccinated group. Thesefindings thus demonstrate that lymphatic pathology was minimal or wasabsent in vaccinated and challenged macaques that did not show Mf intheir peripheral blood.

Example 12: Prophylactic Vaccine Against Dirofilaria immitis Infectionin Dogs

Immunization Protocol.

Six dogs were divided into two groups of three animals per group. Eachanimal of the first group received three rounds of 100 μg dose ofrBmHAXT vaccine plus 400 μl of AL019 adjuvant (40 μg TLR4 agonist+800 μgalum) s/c. Each animal of the second group was used as a control andreceived three rounds of 400 μl of AL019 adjuvant only s/c. Injectionswere performed on days 0, 30, and 60. In addition, at days 0, 30, 60,and 90, 20 mL of blood was collected in EDTA tubes from the saphenousvein of each dog prior to immunization. All animals were monitored foradverse reactions including injection site reactions, fever, loss ofappetite, allergy, hair loss and weight loss.

Antigen-Specific Antibody Levels in Dog Sera.

Levels of rBmHAXT-specific total IgG, IgG1, IgG2, IgG3, IgM and IgEantibodies were determined in the sera of each dog using an indirectELISA as described elsewhere herein.

Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Assay.

ADCC assay was performed as described elsewhere herein. Eight to tenlive D. immitis L3 each were incubated at 37° C. with 5% CO₂ induplicate wells along with 0.5 million PBMC isolated from normal dogblood, 200 μl RPMI 1640 medium and 100 μl of sera samples. Plates weremonitored under light microscope every 24 hours for viability of D.immitis L3. Larvae that were limpid, non-motile or slowly motile wereconsidered dead. The percentage larval death was expressed as the ratioof the number of dead L3 to the total number recovered from each wellmultiplied by 100.

Immunization with rBmHAXT Vaccine Generated High Titer ofAntigen-Specific IgG Antibodies and their Isotypes.

Immunization of dogs with the purified rBmHAXT protein generated hightiter of antigen-specific IgG antibodies (over 1:20000). There was alsoa significant increase in IgG1, IgG2 and IgA antibodies in the serum ofdogs after three immunizations compared to the control group, with IgG1and IgG2 being the most predominant isotype of IgG antibodies. Analysisof the sera samples for the presence of protective antibodies against D.immitis infective larvae showed that significant levels of protectivememory antibodies were present in the sera of vaccinated animals. Theseprotective antibodies proliferated in response to rBmHAXT and were ableto kill both drug-sensitive (˜60%) and drug-resistant (85%) of infectivelarvae after two to three immunizations (FIG. 6) Several cells werefound attached to the dead larvae. Further, culture supernatants ofspleen cells from the vaccinated dogs showed elevated levels of TNF-αand IL-10. These findings suggest that rBmHAXT is an excellent vaccinecandidate against heartworm infections in animals such as dogs and cats.

Example 13: Comparison of rBmHAXT and rDiHAX Vaccines AgainstDirofilaria immitis Infection in Mice

Cloning, Expression and Purification of rBmHAXT Recombinant Protein.

GenScript (Piscataway, N.J.) supplied the sequences of bmhspl2.6(GENBNAK Accession No. AY692227.1), bmalt-2 (GENBNAK Accession No.JF795950.1), bmtpx-2 (GENBNAK Accession No. AF319997.1) and bmtsp(GENBNAK Accession No. JF795955.1) in the pUC57 vector. The genes wereamplified using forward 5′-CGG GAT CCA TGG AAG AAA AGG TAG TG-3′ (SEQ IDNO:31) and reverse 5′-CCC GAA TTC TTA ATG TTT CTC AAA ATA TGC TTT-3′(SEQ ID NO:89) with restriction sites for BamHI and EcoRI. ThePCR-amplified products were cloned into the pRSETA expression vector,transformed into competent BL21 (DE3) E. coli cells for expression ofthe recombinant proteins with 6× histidine tag. Recombinant fusionproteins were purified using immobilized metal affinity Ni⁺-chargedagarose chromatography column sold under the tradename SEPHAROSE® (GEHealthcare Life Sciences, Pittsburgh, Pa.) and eluted with 300 mMimidazole. Endotoxin in the final purified protein preparation wasremoved using an endotoxin removal column (Thermo Fisher Scientific,Rockford, Ill.). The expression and purity of recombinant proteins wasconfirmed in 12% SDS-PAGE gel and western blot using anti-His antibodies(Qiagen, Valencia, Calif.). Protein concentration was determined using aBradford reagent (Thermo Fisher Scientific).

Cloning, Expression and Purification of rDiHAX Recombinant Protein.

The contig nucleotide sequence of DiHSP 12.6 (gene=nDi.2.2.2.g00663),DiALT-2 (gene=nDi.2.2.2.g08197) and DiTPX-2 (gene=nDi.2.2.2.g06574) wereobtained from Wormbase ParaSite by blasting BmHSP 12.6, BmALT-2 andBmTPX-2 sequences. BmHSP 12.6, BmALT-2 and BmTPX-2 proteins were foundto share 97%, 63% and 96% sequence similarity with DiHSP 12.6, DiALT-2and DiTPX-2 proteins, respectively. The nucleotide and amino acidsequence of DiHSP are provided in SEQ ID NO:90 and SEQ ID NO:91,respectively. The nucleotide and amino acid sequence of DiALT-2 areprovided in SEQ ID NO:92 and SEQ ID NO:93, respectively. The nucleotideand amino acid sequence of DiTPX are provided in SEQ ID NO:94 and SEQ IDNO:95, respectively. The nucleotide sequences of DiHSP 12.6, DiALT-2 andDiTPX-2 were linearly combined and the resulting dihax gene wassynthesized by Invitrogen Life Technologies™. The chimeric gene (SEQ IDNO:96) was provided in pET100/D-TOPO® vector (ThermoFisher Scientific,Rockford, Ill.), transformed into competent BL21*DE3 E. coli and therDiHAX fusion protein (SEQ ID NO:97) was expressed. Briefly, anovernight seed culture was inoculated into 500 mL sterile LB broth andallowed to grow under the optimized conditions and selection pressureuntil the OD₆₀₀ was reached. The bacterial cells were then induced with1 mM IPTG (Research Products International, Mt. Prospect, Ill.) andallowed to grow for an additional 4 hours. The cells were then pelleteddown by centrifuging at 12,000 rpm for 30 minutes at 4° C. For proteinpurification, the pellet was re-suspended in 20 mL Tris-Buffered Saline(TBS) and 150 μl of lysozyme was then added to the solution andincubated for 30 minutes in a shaker platform at room temperature.Following incubation, the pellet was sonicated for 4 cycles at 1 minuteeach with a pause of 30 seconds in between. Following sonication, thelysates were centrifuged at 12,000 rpm for 30 minutes at 4° C. Thesupernatant was discarded, the pellet was washed and 15 mL of 8M Ureawas then added to the pellet and incubated overnight at 4° C. withconstant mixing. After incubation, the lysate was centrifuged at 12,000rpm for 30 minutes at 4° C. and the supernatant was collected into afresh 50 mL conical centrifuge tube over ice. The recombinant proteinwas expressed with an N-terminal six histidine residue tag. This allowedfor purification of the rDiHAX protein by Immobilized Metal AffinityChromatography (IMAC). In particular, the extracted protein wasincubated with 2 mL cobalt resin for 30 minutes in a shaker at roomtemperature and packed into a 10 ml column. After washing the columnwith 10 ml TBS, the column was washed with 30 mL of 10 mM Imidazoleprepared in TBS. The bound protein was then eluted with 300 mM Imidazolecontaining 10% glycerol in TBS. Purity and molecular size of the rDiHAXprotein was assessed on a 14% SDS-PAGE gel. The molecular weight of therecombinant protein was approximately 60 kDa. After desalting, endotoxinfrom the purified protein was removed by passing the protein solutionthrough a High Capacity Endotoxin removal resin column (ThermoFisherScientific) The level of endotoxin in the concentrated protein samplewas analyzed using a Pierce™ LAL Chromogenic Endotoxin Quantitation Kit.The final amount of endotoxin in the purified rDiHAX preparation was 3EU per mg of protein.

Experimental Design.

Balb/c mice (male 4-6 weeks of age) were grouped into 10 mice per groupand immunized subcutaneously with 15 μg of rBmHAXT or rDiHAX antigenalong with 10 μg of AL019 (Alum plus GLA, a synthetic TLR4 agonist) asadjuvant. Four immunizations were given at 2-week intervals. Controlanimals received AL019 adjuvant only. Blood samples were collected fromeach mouse prior to each immunization and 2 weeks after the lastimmunization to analyze the serum levels of antigen-specific IgG, IgG1,IgG2a, IgG2b and IgG3. An ADCC assay was performed by incubating 10-15D. immitis L3 with 50 μl of sera from immunized mice and 1×10⁵peritoneal cells from control mice. A challenge experiment was alsoperformed by placing a micropore chamber containing 15-20 D. immitis L3in the peritoneal cavity of all mice. Larval viability was determined 72hours post-challenge and spleen cells and peritoneal fluid/cells wereanalyzed for immunological correlates of vaccine-induced protection.

Immunization with rBmHAXT and rDiHAX Vaccines Generated High Titer ofAntigen-Specific IgG Antibodies and their Isotypes.

Immunization of mice with the purified rBmHAXT and rDiHAX proteinsgenerated high titer of antigen-specific IgG antibodies. In particular,the results showed that immunized animals developed high titers (1:10000titer) of IgG1, IgG2a, IgG2b and IgG3 antibodies against rBmHAXT orrDiHAX compared to controls (p<0.05).

Results from the ADCC experiments showed that sera samples from rBmHAXTimmunized mice killed 93±8.83% larvae and sera samples from rDiHAXimmunized mice killed 76±5.69% larvae compared to the sera samples fromthe AL019 group that gave (20±5.93%) larval death. In the challengeexperiment, larval death was 83±4.14% and 71±8.99% for rBmHAXT andrDiHAX immunized mice, respectively, compared to the control(7.3±2.42%). Notably, there was significant (p<0.05) cross-reactivity ofantibodies in the sera samples from rBmHAXT and rDiHAX vaccinatedanimals with rBmHAXT and rDiHAX proteins as determined by ELISA andwestern blot analysis.

Analysis of the cellular immune response showed that there was anincrease in the antigen-specific CD3⁺CD62L⁺CCR7⁺ memory T cells in thespleen of vaccinated animals compared to AL019 controls. The culturesupernatants of spleen cells from both rBmHAXT and rDiHAX groups showedelevated levels of IL-17A, IL-6, IFN-γ and IL-10.

What is claimed is:
 1. A multivalent immunogenic composition comprisingtwo or more antigens from one or more filarial nematodes.
 2. Themultivalent immunogenic composition of claim 1, wherein the filarialnematodes are selected from the group of Brugia malayi, Wuchereriabancroft, Onchocerca volvulus, Loa loa, Brugia timori and Dirofilariaimmitis.
 3. The multivalent immunogenic composition of claim 1, whereinthe antigens are protein-based, DNA-based, or a combination thereof. 4.The multivalent immunogenic composition of claim 1, wherein the antigenscomprise an Abundant Larval Transcript, Tetraspanin, Small heat shockprotein (HSP) 12.6, Thioredoxin Peroxidase 2, or fragments thereof. 5.The multivalent immunogenic composition of claim 4, wherein the AbundantLarval Transcript comprises SEQ ID NO:121 or SEQ ID NO:122; the Smallheat shock protein 12.6 comprises SEQ ID NO:81 or SEQ ID NO:123; theTetraspanin comprises SEQ ID NO:82; and the Thioredoxin Peroxidase 2comprises SEQ ID NO:83 or SEQ ID NO:124.
 6. The multivalent immunogeniccomposition of claim 1, wherein the antigens are covalently attached. 7.A recombinant vector comprising nucleic acids encoding the multivalentimmunogenic composition of claim
 1. 8. A recombinant host cellcomprising the recombinant vector of claim
 7. 9. The multivalentimmunogenic composition of claim 1 further comprising an adjuvant.
 10. Amethod for inducing a protective immune response in a subject comprisingadministering the multivalent immunogenic composition of claim 1 to asubject thereby inducing a protective immune response in the subject.11. The method of claim 10, further comprising administering one or moreadditional doses of the multivalent immunogenic composition to thesubject.
 12. The method of claim 10, wherein the immunogenic compositionis administered by subcutaneous or intramuscular injection.
 13. Themethod of claim 10, wherein the multivalent immunogenic composition isadministered with an adjuvant.
 14. A method for immunizing an animalagainst filariasis or dirofilariasis comprising administering amultivalent immunogenic composition of claim 1 to a subject therebyimmunizing the subject against filariasis or dirofilariasis.
 15. Themethod of claim 14, further comprising administering one or moreadditional doses of the multivalent immunogenic composition to thesubject.
 16. The method of claim 14, wherein the immunogenic compositionis administered by subcutaneous or intramuscular injection.
 17. Themethod of claim 14, wherein the multivalent immunogenic composition isadministered with an adjuvant.